Morphophysiological Characteristics of Two Duckweed Species Grown in Gold Mine Tailings and HAuCl4 for Phytomining Purpose

Morphophysiological Characteristics of Two Duckweed Species Grown in Gold Mine Tailings and HAuCl4 for Phytomining Purpose | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Morphophysiological Characteristics of Two Duckweed Species Grown in Gold Mine Tailings and HAuCl 4 for Phytomining Purpose Rizki Maulana Yusuf, Aurora Karina Chandra, Miftahudin Miftahudin, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9578120/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Background and aims Phytomining can be utilized to extract gold from low-concentration sources like gold mine tailings. Duckweeds (Lemnaceae) have been known as heavy metal hyperaccumulators. This research aimed to observe the morphological, anatomical, and physiological characteristics of two duckweed species ( Landoltia punctata and Lemna aequinoctialis ) grown in media containing tailings and gold solution (HAuCl 4 ) and their gold accumulating capability. Methods The experiment was carried out by exposing L. punctata and L. aequinoctialis to 2.5 L of Hoagland’s solution treated by 0 g (0%), 100 g (4%), 250 g (10%), 500 g (20%) of tailings suspended by aqua regia, or by 0.7 mM of HAuCl 4 (Au 138 ppm). The treatments and observation were carried out in greenhouse conditions for 7 days. The duckweeds were then harvested for anatomical and physiological measurement, and gold accumulation analysis. Results high content of tailing or Au treatment caused stunting growth in both duckweeds. Anatomical and physiological observations revealed that both duckweeds experienced heavy metal stress, indicated by increasing reactive oxygen species (ROS) and thinner fronds. L. punctata had superior ability to tolerate the stress based on the ability to increase ROS scavengers to countermeasure ROS production. In addition, under the highest tailing content (20% tailing), L. punctata was still able to accumulate gold up to 1.7 µg g − 1 and produced larger gold nanoparticles with gold content reached 2.3% in media containing Au 138 ppm. Conclusions The gold phytomining process caused stress in duckweeds. Nevertheless, both species have great potential as gold phytomining agents. gold recovery heavy metal Lemnaceae phytoremediation sustainable mining Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 INTRODUCTION Gold is a precious and useful metal with high economic value and has been increasing all the time due to high demand. Mining activity is the primary source of gold, but with the limited availability of high-grade gold ore make people search for another alternative source. Secondary gold source is residual materials containing recoverable gold (Syed 2012 ). One such source is gold mine tailings, the waste generated by gold mining activities. Gold mine tailings has a good prospect as an alternative gold source with the fact that tailings are produce almost 2 billion metric ton annually (Oberle et al. 2020 ). However, because of low content of gold and high content of other heavy metals, recovering gold from tailings is considered as economically unprofitable with current method of extraction (Cairncross and Tadie 2022 ). Therefore, efforts to recover gold from tailings effectively, safely, and affordably remain challenging. Phytomining is a method for extracting valuable metals using hyperaccumulator plants that can accumulate these valuable metals and are harvested as a bio-ore which is more environmentally friendly to process (Sheoran et al. 2013 ). Phytomining can become a sustainable method to extract gold from tailings, because this method is considered relatively cheap, easy to apply, and safe from environmental damage. However, this method tends only to be able to accumulate gold in small quantities and need a lot of time for the extraction process (Septian et al. 2025). According to Dinh et al. ( 2022 ), gold phytomining is only considered economically profitable if the gold content in the plant biomass is more than 0.06 mg kg -1 dry weight. Therefore, it is necessary to search for gold hyperaccumulator plant species. Another challenge in phytomining is that growing the plants in gold mine tailings can cause them to undergo stress due to the presence of hazardous materials in mining waste, especially high content of heavy metals (Hilmi et al. 2018 ; Septian et al. 2025). Heavy metals in growing medium can cause stress in terrestrial as well as aquatic plants, and even many aquatic protists, due to disruption of photosynthesis (Ardipeni et al. 2025 ; Hu et al. 2023 ; Septian et al. 2025; Szabó et al. 2023 ), which reduce growth. In phytomining activities, the excessive stress can reduce photosynthesis and the energy supply to plants, which inhibits growth and reduces the ability to accumulate metals (Manzoor et al. 2018 ; Taiz et al. 2015 ; Zhou et al. 2022 ). Changes in morphology, anatomy, and physiology may indicate stress conditions in plants, or be part of their response to increase their ability to survive during unfavorable conditions. The reduction in growth and changes in plant morphology and physiology during the phytomining process emphasize that the plants which are utilized in a phytomining process must have resistance to heavy metals so that they can absorb as much precious metal as possible before the plants are harvested. Duckweeds are fast growth aquatic plants from Lemnaceae family, several species of which, are considered as a hyper accumulator for heavy metals (Sasmaz et al. 2019 ; Seifi and Dehghani 2021 ) and have been used in phytoremediation (Muthan et al. 2024 ; Szabó et al. 2023 ). However, the experiments utilizing duckweed for phytomining purposes, especially for gold, are still rarely found. Our previous study confirmed that a duckweed species ( Lemna aequinoctialis ) was able to extract gold up to 3.8 mg kg -1 in their biomass from gold mine tailings with the addition of aqua regia solution (Yusuf et al. 2025 ). Aqua regia itself acts as gold lixiviant which can boost gold phytomining by increase the solubility of gold and make it available for plants (Anderson et al. 2013 ). Although lixiviant can increase the ability of plants to accumulate gold, its addition usually has a negative impact on plants, because it increases the solubility of other heavy metals in the medium, thus poisoning the plants and reducing their potential to absorb more gold (Krisnayanti et al. 2016 ; Yusuf et al. 2025 ). Therefore, it is necessary to obtain genotypes that are resistant to high levels of heavy metals. Duckweed family have five species which have differences in size and growth rate, so they have the potential to be further studied to explore their capability as phytomining agent. The purpose of this experiment was to observe the adjustment in morphology, anatomy, and physiology of two duckweed species ( Landoltia punctata and Lemna aequinoctialis ) during phytomining and their capability to extract gold from media contained gold mine tailings as well as gold solution in the form of HAuCl 4 . MATERIALS AND METHODS Preparation and acclimatization of duckweeds Two species of duckweeds were used in this experiment which obtained from local aquatic plant farmer. Based on the identification key developed by Bog et al. ( 2020 ), the duckweeds species were Landoltia punctata and Lemna aequinoctialis (Fig. 1 ). These duckweeds were grown and acclimatized in separate plastic tanks (28 cm x 23 cm x 9 cm) contained 2.5 L of Hoagland's full-strength solution which were aerated and placed in a greenhouse. The plants were ready for the treatment after 14 days of acclimatization. The composition of Hoagland’s full-strength solution media followed Epstein and Bloom ( 2005 ) which was composed of 6 mM of KNO 3 ; 4 mM of Ca(NO 3 ) 2 ·4H 2 O; 2 mM of [NH 4 ]H 2 PO 4 ; 1 mM of MgSO 4 ·7H 2 O; 64 µM of Na[Fe(DTPA)]; 50 µM of KCl; 25 µM of H 3 BO 3 ; 2 µM of MnSO 4 ·H 2 O; 2 µM of ZnSO 4 ·7H 2 O; 0.5 µM of CuSO 4 ·5H 2 O; and 0.5 µM of H 2 MoO 4 . All nutrients were dissolved in distilled water. Leaching mineral of gold mine tailings The preparation and leaching of gold mine tailings used in the experiment was based on the method from our previous study (Yusuf et al. 2025 ). Gold mine tailings were obtained from tailings dam facility of Indonesian Gold Company, Aneka Tambang (ANTAM) Inc., UBPE Pongkor, Bogor, West Java, Indonesia (6° 38′ 45″ S, 106° 34′ 20″ E). The leaching process was performed by slowly mixing 4 kg of dry fine tailings with 2 L of aqua regia solution until it evenly mixed and forms paste-like tailings suspension and allowed to stand for 24 hours. After 24 hours, 5% dolomite suspension was used to increase the pH level of tailings suspension up to 5.0 and then allowed to stand for another 24 hours. Preparation of gold solution The gold(III) chloride (HAuCl 4 ) stock solution with a concentration of 0.5 M was prepared by dissolving 2 g of pure gold in 18.2 mL aqua regia solution following the procedure made by Mayoral et al. ( 2014 ). The mixture was left for 48 hours for residual acids removal, then mixed in Hoagland’s media solution and used as a positive control to observe the ability of duckweed to accumulate pure gold ion from the culture media. Gold phytomining process by duckweeds After 14 days of acclimatization in Hoagland’s media, the duckweeds then were grown in the media contained gold mine tailings or gold solution for phytomining experiment. The experiment was carried out following the method developed previously by Yusuf et al. ( 2025 ) with some modification. The experiment was prepared using Completely Randomized Design with three experimental repetition and two variables: duckweed species ( L. punctata and L. aequinoctialis ) and treatment media. There were five media treatments that used in this experiment. The first, second, third, dan fourth treatment were tailings treatment media with the concentration of 0% (negative control), 4%, 10%, and 20% of tailings suspended to Hoagland’s full-strength solution. The tailings treatment medium was prepared by mixing 0, 100, 250, and 500 g of tailings suspension with Hoagland’s media until it reached a volume of 2.5 L. The fifth treatment was Hoagland’s full-strength solution with the addition of HAuCl 4 solution at 0.7 mM labeled as Au 138 ppm medium that was used as positive control to study the responses of duckweeds to gold (Au) ion. Au 138 ppm medium was prepared by diluting 3.26 mL of HAuCl 4 stock solution with Hoagland’s full-strength solution until it reached a volume of 2.5 L. Before duckweed planting, all the treatment were aerated for 48 hours and then the pH of all treatment was adjusted to became 6.0 using dolomite. A 50 g of acclimatized duckweeds were weighed and then were grown and maintained into each treatment medium for seven days. All treatment media were plastic tanks (28 cm x 23 cm x 9 cm) equipped with a plastic cup (with diameter 5.5 cm and height 9 cm) that was glued on plastic tank for specific measurement. The plastic cup was perforated for circulation of media between the tank and the cup. Four duckweed plants with similar size and number frond were placed in the plastic cup that was already glued in the plastic tank for monitoring their growth and morphology changes. Growth and morphology monitoring The frond number of duckweeds grown in the plastic cup placed on each treatment was counted every day for 7 days to calculate the growth. Relative growth rate (RGR) and doubling time (DT) per day were calculated from the number of fronds (NF) in day 1 and day 7 using Eqs. ( 1 ) and ( 2 ) respectively (Ziegler et al. 2015 ). After 7 days of treatment, the picture of duckweed frond on each treatment media was taken using a camera with the same distance to monitor morphological changes. The pictures were then given a scale bar using ImageJ and the mother frond area of each duckweed plant was calculated using freehand tool of ImageJ with two repeated measurements. $$\:RGR=\frac{\text{ln}NF\:day\:7-\text{ln}NF\:day\:1}{day\:7-\:day\:1}$$ 1 $$\:DT=\frac{\text{ln}2\:}{RGR}$$ 2 Harvesting and preparation of duckweed samples After 7 days planting, duckweeds were harvested, cleaned with running distilled water and then drained using paper towel. After dry, the duckweed was weighed to measure the harvested wet weight. For physiological test samples, 10 g of fresh duckweed from each treatment was drenched in liquid nitrogen and keep in a cooling box before the samples were stored in -80°C freezer to keep them fresh. The duckweed from plastic cup was also cleaned and then was fixed for anatomy observation using Formaldehyde Alcohol Acetic Acid (FAA) solution for 24 hours. After 24 hours, the sample then was drenched three times in 50% ethanol solution for 15 mins each and then was stored in 70% ethanol solution. The rest of the sample which was not used for anatomy or physiology analysis will be used for the measurement of water content and gold content analysis. Anatomy observation of duckweed Transverse cross-section of duckweed frond was prepared using paraffin embedded cross-section method (Johansen 1940 ). After it was stored in 70% ethanol solution, the fronds then were dehydrated using a series of Johansen solutions I-VII as dehydrants (Johansen 1940 ). After that, dehydrated fronds were embedded in paraffin block and then were drenched in Gifford solution to soften the sample (Gifford 1950 ). The embedded samples were then sliced transversally using rotary microtome (Yamato RV-240) with thickness of 10 µm. The paraffin from the frond cross-section then washed using xylene and then the cross-section was observed using bright field microscope (Olympus Cx23 with Indomicro camera). The picture of transversal cross-section was captured from the left side and the right side of midrib of frond with three replications for each treatment. The pictures then were analyzed using ImageJ with two repeated measurement which the parameters that were analyzed including frond thickness, epidermis thickness (adaxial and abaxial), mesophyll tissue thickness (MT), mesophyll tissue area, aerenchyma area, and mesophyll cells perimeter (representative) (Pmc), and number of mesophyll cells (Nmc). Aerenchyma fraction of mesophyll tissue (AFM) and the surface area of mesophyll cells per leaf area (Ames/A) was calculated using equations ( 3 ) and ( 4 ) from Sack et al. ( 2013 ) respectively with modification which in the Ames/A calculation, Pmc was based on the average of 10 representative cells which chosen randomly. There were 2 group of representative cells which were small cells group (Pmc < 100 µm) and large cells group (Pmc ≥ 100 µm). Mesophyll cells were modeled as spheres for estimated mesophyll cell surface area (SAmc) and mesophyll cell volume (Vmc) (Eqs. 5 and 6 ). $$\:AFM=\:\frac{\sum\:Arenchyma\:tissue\:area}{Mesophyll\:tissue\:area\:}$$ 3 $$\:\text{A}\text{m}\text{e}\text{s}/\text{A}=\:\frac{\sum\:SAmc\times\:\left(MT\left(1-AFM\right)\right)}{\sum\:Vmc\:}$$ 4 $$\:SAmc=\left(4\pi\:\times\:{\left(\frac{Pmc}{2\pi\:}\right)}^{2}\right)\times\:Nmc$$ 5 $$\:Vmc=\left(\frac{4}{3}\pi\:\:\times\:{\left(\frac{Pmc}{2\pi\:}\right)}^{3}\right)\:\times\:Nmc$$ 6 Physiological analysis of duckweed Physiological aspect of duckweed which were analyzed including H 2 O 2 content, photosynthetic pigment content, ascorbic acid content, total phenolic content, reducing sugar content, and proline content. All the samples were in fresh condition and crushed with addition of liquid nitrogen. H 2 O 2 content was measured using a method from Junglee et al. ( 2014 ) with KI reagent and then the absorbance of sample extract was measured using a UV-vis spectrophotometer (Metertech SP-8001) in 285 nm wavelength. Photosynthetic pigment content which consisted of chlorophyll a, chlorophyll b, and total carotenoids were measured using method from previous study (Wellburn 1994 ) which the pigment was extracted using 80% acetone solution and then the absorbance of extract was measured in wavelengths of 470, 646, and 663 nm. The equations from the same studies were used to calculate the content of chlorophyll a, chlorophyll b, and total carotenoids which can be seen in Eq. 7 , 8 , 9 respectively. Ascorbic acid from the plant was measured using Abera et al. ( 2020 ) method which the plant extract was reacted with 0.335 mM K 2 Cr 2 O 7 and 0.185 mM MnCl 2 and then the absorbance was measured twice immediately at 350 nm and later after 5 mins delay. The method from Ghanem et al. ( 2021 ) was used to measure the total phenolic content which the plant extract was reacted with Folin Ciocalteu reagent and then the absorbance was measured at 765 nm. Reducing sugar content from duckweed was measured using a method from Khatri and Chhetri ( 2020 ) which hot 3,5-Dinitrosalicylic acid reagent was reacted with plant extract and then the absorbance was measured at 540 nm. Proline content was measured using method from Schweet ( 1954 ) which the plant extract was reacted with Acid Ninhydrin reagent to produce chromogen then was extracted using toluene and the absorbance of chromogen was measured at 520 nm. All the physiological measurement was done twice. $$\:Chlorophyll\:a=12.25\left(Absorbance\:663\:nm\right)-2.79\left(Absorbance\:646\:nm\right)$$ 7 $$\:Chlorophyll\:b\:=21.5\left(Absorbance\:646\:nm\right)-2.79\left(Absorbance\:663\:nm\right)$$ 8 $$\:Total\:carotenoid=\frac{1000\left(Absorbance\:470\:nm\right)-1.82\left(Chlorophyll\:a\right)-85.02\left(Chlorophyll\:b\right)}{198}$$ 9 Plant dry weight measurement The fresh weight of sample from each treatment was measured then the sample was dried in oven at 60°C for 72 hours. After dried, the sample weight was measured again as dry weight. The water content of duckweed was measured base on the changes of plant weight before and after drying process (Eq. 10 ). Harvest dry weight of duckweed from each treatment media was estimate based on harvest wet weight and water content (Eq. 11 ). $$\:Water\:content=\frac{\left(wet\:weight-dry\:weight\right)}{wet\:weight}\:\times\:100\%$$ 10 $$\:Harvested\:biomass=harvested\:wet\:weight\:\times\:\left(100\%-water\:content\right)$$ 11 Gold content analysis and phytomining aspect analysis The gold content analysis in duckweed biomass and tailings followed the method from Satya et al. ( 2018 ) with modification to adjust with gold sample. The analysis was started with the destruction of 0.1 g of dry sample in digestion vessel with mixed solution of 3 mL of 30% H 2 O 2 , 3 mL of concentrated HNO 3 , and 5 mL of deionized water then the mixture was heated using an autoclave at a temperature of 121°C with a pressure of 1 atm for 45 mins. After heated, the pH of mixture was raised to 2 by adding 1 N KOH solution and then 2 mL of 6 M KSCN solution was added into the mixture and allowed to stand for 24 hours to dissolve the remaining gold. After 24 hours, the mixture was added again with 1 mL of 30% H 2 O 2 and 1 mL of concentrated HNO 3 and then was heated again in the autoclave with the same parameter as before to obtain a clear digestate. The gold content from digestate was then measured using a graphite furnace atomic absorption spectrophotometer (GF-AAS) (Hitachi Z 2700). The potential of duckweed used as gold phytomining agent was looked from bio-concentration factor (BCF) Bioconcentration factor was calculated following the equation from Verma & Suthar ( 2015 ) which a ratio between gold content in plant with gold concentration in media (Eq. 12 ). Gold concentration for tailings treatment media was estimated from the amount of tailings in the media and the tailings gold content. $$\:BCF=\frac{Gold\:content\:in\:biomass\:}{Initial\:gold\:concentration\:in\:media}$$ 12 Statistical analysis The statistical test two-way ANOVA with p -value < 0.05 was used in this research unless it was stated using different statistical test. Significantly different tests resulted from ANOVA were then analyzed further using Tukey HSD analysis. The statistical analyses were done in IBM SPSS Statistic 25. RESULTS Gold mine tailings and gold solution decreased the growth of duckweeds The growth of duckweed measured based on frond number, relative growth rate and doubling growth was influenced strongly by gold mine tailings as well as gold solution. The changes of fronds number in both duckweed species during 7 days treatment can be seen in Fig. 2 . In media without addition of tailings or gold solution (0% media), the growth of frond number was faster on L. aequinoctialis compared to L. punctata. The ability of L. aequinoctialis to grow faster compared to L. punctata also can be seen in media with high tailings (20%) or gold content (Au 138 ppm) which there was a slight growth on number of fronds each day for L. aequinoctialis while the number of fronds of L. punctata did not change after day 2 until day 7 of treatment (Fig. 2 ). Other growth parameters (relative growth rate and doubling time) also had similar trend (Fig. 3 ). The relative growth rate of L. aequinoctialis was significantly higher than L. punctata in 0% tailings media. The relative growth rate of these two plants was decline along with the increase of tailing content in media. The 138 ppm gold in Hoagland’s media (Au 138 ppm) also caused a significant decrease on relative growth rate in both plants. Lastly, doubling time for L. aequinoctialis did not show a significant difference between each treatment while L. punctata in 20% tailing and Au 138 ppm media had significantly longer doubling time compared to negative control media. Morphological responses of duckweed during gold phytomining During 7 days of treatment using 4, 10 and 20% of gold mine tailing and Au solution, the plants underwent morphological changes as compared to control plants (Fig. 4 ). The color of L. punctata fronds after 7 days treatment in media that consisted of 0%, 4%, 10%, and 20% shifted from green into pale-yellow (Fig. 4 a, b, c, and d). The same changes on fronds color between tailing treatment media also happened to L. aequinoctialis (Fig. 4 f, g, h, and i). In contrast, the frond color of these two species was turning into purple after grew in Au 138 ppm media for 7 days. The area of mother fronds of L. punctata and L. aequinoctialis were not significantly different for all treatments media for each duckweed species (Fig. 5 ). Frond anatomy changes of duckweeds during gold phytomining The transverse cross-section of frond from both duckweed species suggested that gold mine tailings and Au solution treatments reduced the thickness of frond anatomy on both species, especially due to the reduction of mesophyll cells due to the treatments, except on L. punctata grown at 10% of gold mine tailing (Figs. 6 and 7 ). Frond thickness of L. punctata in 10% tailings treatment media was the highest compared to other treatments (Fig. 7 a) and it can be associated with their mesophyll tissue thickness which had the same pattern (Fig. 7 b). However, for L. aequinoctialis which grew in tailings treatment media, the thickness of frond and mesophyll were decreasing with the increase of tailing content in media. The thickness of adaxial epidermis from L. aequinoctialis did not have a significant difference between all treatment media while for L. punctata which grew in 10 and 20% tailings media had significantly thicker adaxial epidermis compared to 0% tailings media (Fig. 7 c). The cross-section of these two duckweeds frond which grew in Au 138 ppm media showed that this treatment caused the thickness of frond and mesophyll tissue thinner compared to their respective negative control (0% tailings treatment). Furthermore, this treatment also caused the cells of these duckweed fronds turning color into purple with the lower part of frond (abaxial) had darker coloration compared to upper part (adaxial). For abaxial epidermis (Fig. 7 d), L. punctata that grew in 20% tailings treatment media had thicker abaxial epidermis compared with other treatment while for L. aequinoctialis , the plant that had the thickest abaxial epidermis was L. aequinoctialis in 0% tailing treatment media. The fraction of aerenchyma in mesophyll tissue of duckweeds frond can be seen in Fig. 7 e. The fraction of aerenchyma of L. punctata in all treatment media did not have significant different from each other. For L. aequinoctialis , the plant that grew in 10% tailing treatment media had significantly large total area of aerenchyma in their mesophyll tissue compared to other treatment. for Ames/A of L. punctata , all plant in tailings treatment media did not have significant different while Ames/A for L. punctata in Au 138 ppm treatment was significantly lower compared to negative control (0% tailings treatment) (Fig. 7 e). The Ames/A of L. aequinoctialis in 0% and 4% tailings treatment media were higher compared to other three treatments. Physiological responses of duckweed during phytomining To understand the physiological responses of the duckweeds, some parameters including the content of H 2 O 2 , photosynthetic pigments, ascorbic acid, reducing sugar, phenolic as well as proline were measured. H 2 O 2 levels in duckweeds after 7 days of treatment can be seen in Fig. 8 a. It can be seen that L. aequinoctialis showed an increase in H 2 O 2 levels in plant grown on media containing tailings (4%, 10%, 20% tailings media) when compared to control negative (0%). Meanwhile, L. punctata did not show a clear pattern with the 4% and 10% tailings treatments, causing a decrease in H 2 O 2 levels, but the 20% tailings treatment was not significantly different from the control. Both duckweed species that grown in the 138 ppm Au treatment showed relatively fewer H 2 O 2 levels than the control. The contents of chlorophyll a, chlorophyll b, and total carotenoids can be seen in Fig. 8 b, c, and d respectively. The content of these three photosynthetic pigments is significantly reduced in both of plants grown in media containing 20% tailings. The response of the two duckweeds after being grown in media containing 138 ppm gold (Au 138 ppm) for seven days showed a decrease in the levels of photosynthetic pigments compared to the control (0% tailings treatment). Ascorbic acid levels in duckweeds after seven days of treatment can be seen in Fig. 8 e. L. punctata grown in 10%, 20% tailings, and Au 138 ppm treatment media showed ascorbic acid levels that were significantly higher than the control. L. aequinoctialis grown in 4% tailings media showed lower ascorbate levels than the control. The reducing sugar levels of duckweeds grown for seven days can be seen in Fig. 8 f. The reducing sugar levels in L. punctata and L. aequinoctialis in the 10% tailings treatment showed the highest levels compared to other tailings treatments. In the 138 ppm Au treatment, both plants showed a decrease in reducing sugar levels compared to the control. Figure 8 g shows duckweeds total phenolic content after seven days of treatment. Total phenolic content of L. punctata showed a significant decrease as the level of tailings added to the media increased. Meanwhile, for L. aequinoctialis , the total phenolic content in each tailings treatment did not show significant differences. Both of plants which grew in 138 ppm Au treatment showed significantly lower levels of total phenolics and flavonoids than the control. Figure 8 h shows the proline levels in duckweeds after seven days of treatment. The proline levels of L. punctata grown in 20% tailings media showed significantly higher proline levels than other treatments. Meanwhile, the proline levels of L. aequinoctialis in each treatment did not show significant differences. Gold accumulation and gold nanoparticles synthesis in duckweeds Figure 9 a shows the accumulation of gold in duckweed biomass after seven days of treatment in tailings. The gold content in plant that grew on 0% tailings media is considered as the lowest gold concentration that generally found in the duckweed (basal content). In 4% tailings treatment media, L. punctata did not show a significant gold accumulation compared to the 0% tailings media treatment while the gold content of L. aequinoctialis in 4% tailings treatment media was significantly higher than their 0% treatment media. Gold accumulation in both plants significantly increased compared to the 0% tailings media treatment when the plants were grown in the 10% tailings media treatment. However, adding 20% tailings suspension to the media did not cause a more significant increase in gold accumulation compared to the 10% tailings treatment in both plants. In the 138 ppm Au treatment, it is known that gold accumulation by L. punctata and L. aequinoctialis can reach 2% of plant biomass (Fig. 9 b). Gold content in this treatment is much higher than that of plants grown in all tailings media treatment. L. punctata on 138 ppm Au treatment media can accumulate gold up to 23.3 mg g -1 on average, which is significantly higher than gold accumulation on L. aequinoctialis (18.0 mg g -1 ). The plants that grew in Au 138 ppm media were extracted of their gold nanoparticles because of high gold accumulation. The color of extract from L. punctata was dark purple while for L. aequinoctialis had purple coloration (Fig. 10 a). Based on absorption spectrum of extracts, it is known that both showed LSPR for spherical gold nanoparticle (peak at wavelength 500–550 nm) with the L. aequinoctialis extract have absorption peak at 538 nm while L. punctata had absorption peak at 545 nm (Fig. 10 b). Phytomining aspects of duckweeds grown in 4, 10, and 20% tailings treatment media can be seen in Table 1 . L. aequinoctialis has a relatively higher harvested wet weight than L. punctata . However, L. punctata has a relatively higher harvested dry weight than L. aequinoctialis . The water content in L. punctata was relatively lower compared to L. aequinoctialis. Tailings sample used in this study had a gold content of 4.78 ± 0.458 µg g -1 , and the gold concentration in tailings treatment media was even smaller due to the dissolution process. The bioconcentration factor (BCF) value of both duckweeds showed a high value (BCF > 1). Based on mass gold in plant, it is known that L. punctata grown in 10% tailings treatment media can produce gold that is higher than other treatments. Table 2 shows the phytomining aspects of the two duckweeds species grown in 138 ppm Au treatment media which containing gold solution (HAuCl 4 ). In this treatment, the wet and dry harvested weight did not show significant differences between the two duckweed species. The water content of the two plants is also similar. The bioconcentration factors of Au from L. punctata and L. aequinoctialis were 179 and 144, respectively. The mass of gold that was successfully harvested from both plants in this medium was much higher than the tailings treatment, with the average of mass gold in plant from L. punctata being higher than L. aequinoctialis Table 1 Aspects of the gold phytomining process in gold mine tailings by duckweeds Aspect Duckweed species L. punctata L. aequinoctialis Tailing content in the media 4% 10% 20% 4% 10% 20% Gold concentration in media (mg L − 1 ) Δ 0.13 0.32 0.64 0.13 0.32 0.64 Gold content in biomass (µg g − 1 ) 0.9 ± 0.09 c 1.6 ± 0.06 ab 1.7 ± 0.19 a 1.3 ± 0.25 bc 1.5 ± 0.11 ab 1.5 ± 0.04 ab BCF 7 5 3 10 5 2 Harvested wet weight (g) 29.6 ± 4.35 b 34.4 ± 3.34 ab 39.8 ± 6.84 ab 46.9 ± 5.04 ab 49.6 ± 10.18 a 42.7 ± 10.26 ab Water content (%) 90 ± 1.7 b 91 ± 1.4 b 94 ± 0.8 a 95 ± 1.1 a 95 ± 1.0 a 96 ± 0.2 a Harvested biomass (g) 3.0 ± 0.44 a 3.1 ± 0.30 a 2.4 ± 0.41 ab 2.4 ± 0.26 ab 2.4 ± 0.50 ab 1.8 ± 0.43 b Mass gold in plant (µg) 2.8 ± 0.41 b 4.8 ± 0.47 a 4.1 ± 0.71 ab 3.0 ± 0.33 b 3.7 ± 0.76 ab 2.6 ± 0.63 b Δ : Media gold content is estimated from the amount of tailings in the media where the gold content in the tailings was 4.78 ± 0.458 µg g -1 . BCF: bioconcentration factor. The values represent the average of 3 replicates. Different superscript letters indicate significant differences from Tukey HSD test at p < 0.05. ±: standard deviation Table 2 Aspects of the gold phytomining process in gold solution (HAuCl 4 ) by duckweeds Aspect Duckweed species L. punctata L. aequinoctialis Au content in the media Au 138 ppm Au 138 ppm Gold content in media (mg L − 1 ) 138 138 Gold content in biomass (mg g − 1 ) 23.3 ± 1.36 a 18.0 ± 2.95 b BCF 169 131 Harvested wet weight (g) 40.2 ± 15.05 a 35.5 ± 6.28 a Water content (%) 94 ± 0.8 a 95 ± 0.2 a Harvested biomass (g) 2.3 ± 0.86 a 1.9 ± 0.33 a Mass gold in plant (mg) 53.5 ± 20.00 a 33.5 ± 5.92 a BCF: bioconcentration factor. The values represent the average of 3 replicates. Different superscript letters indicate significant differences from Tukey HSD test at p < 0.05. ±: standard deviation DISCUSSION Growth and morpho-physiological characteristics of duckweed in phytomining process Duckweed is an aquatic plant belongs to Lemnaceae family (Xu et al. 2021 ) which has very rapid growth rates. In this study, the two duckweeds had different growth rates, i.e.: 3.1 fronds per day for L. punctata and 7.3 fronds per day for L. aequinoctialis in the negative control treatment (Fig. 2 ). However, both species experienced a significant decrease in growth rate when treated with gold mine tailings and Au solution, and the rate of decline was consistent with the increase in tailings concentration (Figs. 2 and 3 ). This indicates that the tailings and Au solution treatments negatively impacted both duckweeds. Based on previous studies by Hilmi et al. ( 2018 ) showed that gold mine tailings collected from an Indonesian gold company, Aneka Tambang Inc. contained several metal and heavy metals with high concentrations including magnesium (Mg): 3962.71 ppm, iron (Fe): 10348.15 ppm and Manganese (Mn): 1791.46 ppm, lead (Pb): 93 ppm, silver (Ag): 13 ppm, and cadmium (Cd): 1 ppm. The use of aqua regia compounds as gold lixiviant in tailings caused an increase in the solubility of heavy metals contained in the tailings (Yusuf et al. 2025 ) so that they were easily absorbed by plants and consequently were toxic to the duckweeds within 7 days of treatment. Cao et al. ( 2025 ) also reported that Lemna minor and L. punctata had similar growth rates when they grew in control media and then it declined significantly with almost the same growth rate when these two duckweeds were grown in 10 mg L -1 of cadmium (Cd) media. Apart from tailings, the study also revealed that Au 3+ solution in the form of HAuCl 4 also significantly hindered the duckweeds growth (Figs. 2 and 3 ). This data is also in accordance with Taylor et al. ( 2014 ) who revealed that K(AuCl 4 ) in concentration of 200 mg L -1 significantly inhibited the growth of Arabidopsis thaliana root. In addition to growth, the level of stress on duckweed due to gold mine tailings and gold solution treatment can also be observed in the change of frond colour of both duckweeds which changed from green to whitish yellow (Fig. 4 ). The colour change was verified by the decrease in the content of photosynthetic pigments, including chlorophyll a , chlorophyll b and carotenoids (Fig. 8 b, c, and d). This decrease in plant pigments was probably due to heavy metal stress from tailings and HAuCl 4 solution treatment. Several studies have also shown that plants experiencing heavy metal stress underwent a decrease in chlorophyll content (Li et al. 2015 ; Zhang et al. 2020 ). Heavy metal treatment can stimulate oxidative stress due to the formation of large amounts of reactive oxygen species (ROS) such as hydrogen peroxide (H 2 O 2 ), superoxide (O 2 - ), and hydroxyl radicals (HO • ) (Hu and Wang 2025 ; Sood 2025 ). In this experiment, only L. aequinoctialis experienced an increase in H 2 O 2 in response to the tailings treatment, while L. punctata did not (Fig. 8 a). This is suspected to be related to the L. punctata genotype's ability to control ROS through a good antioxidant system, as indicated by the increase in ascorbic acid and proline content in L. punctata , especially in the tailings treatment (Fig. 8 e and h). There are 2 hypothesis that could explain the reduced H 2 O 2 content in Au 138 ppm treatment compared to negative control (Fig. 8 a). The first hypothesis is H 2 O 2 content had peaked earlier than the sampling time which it could happen when the plant activated ROS scavenging mechanism after sensing the increased H 2 O 2 content in cells. Kabala et al. (2022) reported that treatment with high concentration of NaCl stimulated the formation of H 2 O 2 , in Cucumis sativus on the first day of treatment, although it decreased again after 6 days of stress. This decrease can be corelated with their catalase activity which increased significantly in 6 days of treatment (Kabala et al. 2022). The second hypothesis is gold nanoparticles in plant cells can catalyzed the decomposition of H 2 O 2 into hydroxyl radicals (HO • ) or O 2 and H 2 O depends on pH (He et al. 2013 ; Liu et al. 2021 ). Ascorbic acid, carotenes, tocopherols, and glutathione are commonly compounds produced by plants in response to environmental stressors (Gupta et al. 2018 ). Ascorbic acid is an important antioxidant for plants in controlling ROS levels (Xiao et al. 2021 ). The reaction between ascorbic acid and free radicals like ROS produces dehydroascorbic acid, which is an oxidized form of ascorbic acid. Jung et al. ( 2019 ) showed that Oryza sativa seedlings grown in media with arsenic (As) did not increase their ascorbic acid, but their ROS scavenging activity may be correlated with the increase in dehydroascorbic acid. In addition, proline, besides has been known as an osmolyte compound in the face of osmotic stress, also plays an important role in controlling ROS in tissues (Renzetti et al. 2025 ). In some species, a slight increase in ROS concentration due to mild stress can act as oxidative signalling, which temporarily increases the ROS scavengers to balance the ROS accumulation (Sood 2025 ). The high ROS and low ascorbate levels in L. aequinoctialis during tailing treatment may indicate a relatively lower resistance level of this species to tailing treatment and may be an indication of why the photosynthetic pigment content in this species was significantly lower than that of L. punctata (Fig. 8 a and e). Another common antioxidant in plants is phenolic which often increases when plants experience stress condition. However, under tailings treatment, the phenolic content of L. punctata decreased as the content of tailings in media increase while L. aequinoctialis did not show any significant change in their phenolic content in all tailings treatment media (Fig. 8 g. Under certain circumstances, severe salinity stress decreased the production of phenolic compound by downregulating phenylalanine ammonia-lyase (PAL) enzyme which involved in phenolic biosynthesis in plants (Mrázová et al. 2017 ). In addition, studies from Pungin et al. ( 2023 ) revealed that adding NaCl up to 100 mM can increase phenolic content of Glaux maritim plant, but then the phenolic content drop when the concentration of NaCl exceeded 100 mM. The reason behind the phenolic content decreasing during severe salinity stress is still unknown but it is believed that plants focus to produce small molecule osmolyte such as proline and simple sugar when the cells osmotic pressure was high. The results of frond anatomy analysis also showed that treatment with tailings and gold solution caused a decrease in frond thickness, especially due to a significant decrease in mesophyll cell thickness in both duckweed species, except for L. punctata in the 10% tailings treatment which experienced an increase (Fig. 7 a and b). The decrease in frond thickness is suspected to be due to ultrastructural damage of frond cells due to the effects of heavy metals, as observed by Baruah et al. ( 2016 ) in the Cd treatment, and Prasetya et al. ( 2022 ) in J. curcas and R. trisperma grown in media containing 100% gold mine tailings. Rucińska-Sobkowiak (2016) also mentioned that the anatomical change in plants caused by heavy metals can be directly from heavy metal toxicity in plant cells or indirectly from abscisic acid (ABA) stress signaling in plants. The increase of mesophyll cells in L. punctata is suspected to be related to hormesis response. Hormesis response is phenomenon where low intensity stressor has beneficial effect for plants (Agathokleous et al. 2023 ). These responses including the increased of frond thickness, mesophyll thickness, ascorbic acid, and reducing sugar showed the level of resistance of this species to stress. Decreased leaf thickness is a common symptom experienced by plants exposed to heavy metal stress. Using 4 species of non-edible oil-producing plants ( Jatropha curcas , Ricinus communis , Melia azedarach and Reutealis trisperma ) Hamim et al. ( 2019 ) found that the treatment using mercury in the form of Hg(NO 3 ) 2 at a concentration of 3 mM caused the decrease of leaves thickness all the species except R. tripserma which indicatied that R. tripserma is more tolerant to Hg than the others. Other factors determined leaf thickness may be related to the ability to absorb water, as found by Yao et al. ( 2023 ) that Lycium barbarum increased their leaf thickness under salt stress to retain water and improve water availability in their leaves. Aside to make duckweeds afloat in water, the frond aerenchyma have crucial function to ensure gas transport in duckweed frond. In these studies, tailings in high concentration caused duckweeds had large aerenchyma in their frond which appears significant in L. aequinoctialis (Fig. 7 e). Kim et al. ( 2024 ) studies revealed that phytohormone ethylene is responsible for the increasing of aerenchyma areas in duckweed ( Spirodela polyrhiza ) by promoting lysigeny via programmed cell death (PCD). Stress including stress by heavy metals could increase the production of ethylene by plants as part of stress response mechanism (Nguyen et al. 2021 ). The area of aerenchyma and mesophyll thickness can influence the photosynthetic capacity by changing the mesophyll surface area (Ames/A) (Sack et al. 2013 ). From previous studies by Longstreth and Nobel ( 1979 ), during salinity stress, plant that reactive to salinity increased their Ames/A by thickening their leaves to conversate stomata closure during stress. Leached tailings containing a lot of soluble solid include heavy metals which can induce salinity and heavy metal stress. Comparing Ames/A from these two species revealed that L. punctata had better mechanism to counter measured stress by tailings as their Ames/A was relatively constant in all tailings treatment media while high concentration of tailings caused Ames/A of L. aequinoctialis decrease significantly (Fig. 7 f). Effectiveness of two duckweed species in gold phytomining Duckweed (Lemnaceae) has been considered as highly resistance aquatic plant to various pollutants in water, including heavy metals (Golob et al. 2021 ; Muthan et al. 2024 ; Szabó et al. 2023 ), which promotes the utilization of duckweed as a heavy metal-absorbing plant for phytoremediation programs. However, there has not been much research that reveals the potential of duckweed in the phytomining of precious metals. The duckweed's ability to absorb large amounts of gold, suggests the potential role of this species in phytomining of gold mine tailings (Yusuf et al. 2025 ). Meanwhile, the use of duckweed in phytomining also faces obstacles considering that gold mine tailings contain several heavy metals in high amounts. Based on gold absorption data, both duckweed species ( L. punctata and L. aequinoctialis ) have the ability to absorb gold very well from gold mine tailings and from gold solution in form HAuCl 4 given in the media, with bioconcentration factor values ​​for gold between 2 and 7, even though the BCF value continued to decrease with increasing tailings concentration. On the other hands, the treatment with addition of HAuCl 4 showed a very high BCF value, i.e.: 131 and 169 for L. aequinoctialis and L. punctata respectively (Tables 1 and 2 ). The BCF values ​​of these two species are much higher than those obtained by Septian et al. (2025) in Amaranthus spinosus and Brassica juncea plants grown in 100% gold mine tailings in combination with ammonium thiocyanate applications. The gold content in both duckweed species was also quite high, i.e.: 0.9–1.7 mg Kg -1 (Table 1 and Fig. 9 a) and has exceeded the minimum concentration required as a plant for phytomining purposes by Dinh et al. ( 2022 ), i.e.: at a minimum of 0.06 mg kg -1 . Among the two duckweed species, gold absorption increased linearly with increasing tailings concentration up to 10%, but only in L. punctata it still increased at the tailings concentration of 20%, while the absorption in L. aequinoctialis it was decreased (Fig. 9 a). This condition is in line with the observed physiological parameters, that L. punctata has higher levels of ascorbate and proline as a sign of a better resistance level than L. aequinoctialis (Fig. 8 e and h). However, because at a tailings concentration of 20%, both species experienced a decrease in biomass, so that the highest total gold was obtained in the 10% tailings concentration treatment (Table 1 ). Furthermore, gold absorption rate in media with HAuCl 4 solution also suggested that L. punctata had significantly higher Au absorption rate (23.3 mg Kg -1 ) than L. aequinoctialis (18 mg Kg -1 ) which indicates a better resistance level (Table 2 ). The decrease in BCF value due to increasing tailings concentration indicates that in addition to the increase of gold content in the media, increasing tailings concentration also means increasing the content of other heavy metals, thereby reducing plant’s ability to absorb gold alone. Meanwhile, High levels of heavy metals cause the plants to experience higher stress so that their absorption ability decreases. As a comparison, using Pb treatment, Manzoor et al. ( 2018 ) found that most of the ornamental plants grown in the highest Pb treatment media (2000 mg kg -1 ) had a lower BCF compared to other treatments with lower Pb concentration. Another interesting aspect of these two duckweed species is the formation of gold nanoparticles, which was clearly observed when both species were treated with a 138 ppm Au solution (HAuCl 4 ). This was indicated by the formation of a purple color on the fronds of both species (Fig. 4 ) and by the purple color of the plant extracts as well as the formation of an absorption peak in the range of 530–550 nm (Fig. 10 a and b). The purple coloration from these two duckweeds did not come from anthocyanin accumulation but from formation of gold nanoparticles inside the plants. This conformed by the purple pigment did not soluble in ethanol solution (used for reducing sugar quantification) which it normally should extract anthocyanins pigment. Our previous studies successfully confirmed the formation of gold nanoparticles in L. aequinoctialis that grew in Au 138 ppm treatment media using transmission electron microscope (TEM) (Yusuf et al. 2025 ). A similar finding was also demonstrated by Taylor et al. ( 2014 ) who found that A. thaliana plants grown in a medium containing gold solution in form of KAuCl 4 changed their leaf color from green to purple, indicating the accumulation of gold nanoparticles in the tissue. Based on the data above (Fig. 10 ), both species produce gold nanoparticles in their tissue after exposing them to media containing HAuCl 4 . The purpose of the 138 ppm Au treatment was to observe the ability of duckweeds to absorb gold without disturbance from other heavy metals. It seems that L. punctata was superior to L. aequinoctialis in accumulating gold in their biomass (Tables 1 and 2 ). Gold nanoparticles from L. punctata are predicted to have larger particle sizes compared to L. aequinoctialis based on the red shift phenomenon (Fig. 10 b), where the LSPR peak of gold nanoparticles shifts to longer wavelengths as the particles increase in size (Kimling et al. 2006 ). The large size of gold nanoparticles in L. punctata may be related to their ability to accumulate gold at high concentrations. In the chemical synthesis of gold nanoparticles, the ratio of gold ion to reductant compound plays a crucial factor in determining the size of nanoparticles, which typically have a larger size when the gold ion is more abundant in the reaction (Shi et al. 2017 ). The superiority of L. punctata in gold absorption was in line with physiological changes that can reduce the stress damages characterized by higher ascorbic acid and proline content compared to L. aequinoctialis . Nevertheless, both have great potential as gold phytomining agents in media containing low gold content, such as tailings. CONCLUSION Two duckweed species ( L. punctata and L. aequinoctialis ) grown in a medium contained gold mine tailings and gold solution (HAuCl 4 ) during the phytomining process experienced stress, resulting in a decrease in growth rate during 7 days of treatment. After the treatment, the frond changed from green to yellow in the tailing’s medium treatment, while the frond of duckweeds grown in HAuCl 4 medium turned color to purple. Both the tailings and HAuCl 4 treatments also caused a significant decrease in the frond thickness of the duckweeds. The phytomining process with tailings and HAuCl 4 induced oxidative stress in duckweed, and in response, the plant produced higher ROS-scavenging compounds including proline and reduced sugar. L. punctata had a higher resistance level to oxidative stress caused by heavy metals compared to L. aequinoctialis based on morphological and physiological parameters. Both types of duckweed can be categorized as good gold phytomining agents for gold mine waste and can accumulate gold up to 1.7 and 1.5 µg g -1 for L. punctata and L. aequinoctialis , respectively. In Hoagland solution containing 138 ppm Au, gold accumulation by L. punctata almost reached 2.3% of their biomass, which was significantly more abundant compared to L. aequinoctialis which was only 1.8%. Gold accumulation by duckweed grown in gold solution (HAuCl 4 ) was also high enough to induce the synthesis of gold nanoparticles indicated by purple color of their fronds, that was verified by LSPR measurements showing the formation of peaks at 545 nm and 538 nm for L. punctata and L. aequinoctialis , respectively. Declarations Competing interests The authors have no relevant financial or non-financial interests to disclose. Funding The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. Author contributions Rizki Maulana Yusuf: investigation; methodology; conceptualization; data curation; formal analysis; project administration; writing – review and editing. Aurora Karina Chandra: investigation; methodology; resources; data curation; visualization; formal analysis; project administration. Hamim: supervision; conceptualization; writing – review and editing; data curation; validation. Miftahudin: supervision; validation; data curation. Dorly: supervision; validation; data curation. Awalina Satya: supervision; validation; data curation. Evi Susanti: supervision; validation; data curation. All authors have read and agreed to the final version of the manuscript. ACKNOWLEDGEMENT The authors gratefully acknowledge the Research Center for Limnology-National Research and Innovation Agency of the Republic of Indonesia (BRIN) for assistance in analyzing our samples. Also, many thanks to PT. Aneka Tambang (ANTAM Inc.) 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J Biol Chem 208(2):603–613. https://doi.org/10.1016/S0021-9258(18)65587-5 Seifi A, Dehghani M (2021) Influence of nano-titanium dioxide particles (TiO2 NPs) on improving phytoremediation efficiency of As/Cu/Cd from copper mine wastewaters using Lemna minor. Arab J Geosci 14(6):494. https://doi.org/10.1007/s12517-021-06842-9 Septian RR, Sulistijorini, Surono H (2025) Dark septate endophytic fungi and thiocyanate induced gold accumulation of Brassica juncea and Amaranthus spinosus grown on gold mine tailings. Sains Malaysiana 54(7):1687–1699. 10.17576/jsm-2025-5407-04 Sheoran V, Sheoran AS, Poonia P (2013) Phytomining of gold: a review. J Geochem Explor 128:42–50. https://doi.org/10.1016/j.gexplo.2013.01.008 Shi L, Buhler E, Boué F, Carn F (2017) How does the size of gold nanoparticles depend on citrate to gold ratio in Turkevich synthesis? Final answer to a debated question. J Colloid Interface Sci 492:191–198. https://doi.org/10.1016/j.jcis.2016.10.065 Sood M (2025) Reactive oxygen species (ROS): plant perspectives on oxidative signalling and biotic stress response. Discover Plants 2(1):187. https://doi.org/10.1007/s44372-025-00275-4 Syed S (2012) Recovery of gold from secondary sources—A review. Hydrometallurgy 115:30–51. https://doi.org/10.1016/j.hydromet.2011.12.012 Szabó S, Zavanyi G, Koleszár G, del Castillo D, Oláh V, Braun M (2023) Phytoremediation, recovery and toxic effects of ionic gadolinium using the free-floating plant Lemna gibba . J Hazard Mater 458:131930. https://doi.org/10.1016/j.jhazmat.2023.131930 Taiz L, Zeiger E, Møller IM, Murphy AS (2015) Plant physiology and development. Sinauer Associates, Massachusetts Taylor AF, Rylott EL, Anderson CW, Bruce NC (2014) Investigating the toxicity, uptake, nanoparticle formation and genetic response of plants to gold. PLoS ONE 9(4):e93793. https://doi.org/10.1371/journal.pone.0093793 Verma R, Suthar S (2015) Lead and cadmium removal from water using duckweed– Lemna gibba L.: Impact of pH and initial metal load. Alexandria Eng J 54(4):1297–1304. https://doi.org/10.1016/j.aej.2015.09.014 Wellburn AR (1994) The spectral determination of chlorophylls a and b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution. J Plant Physiol 144(3):307–313. https://doi.org/10.1016/S0176-1617(11)81192-2 Xiao M, Li Z, Zhu L, Wang J, Zhang B, Zheng F, Zhao B, Zhang H, Wang Y, Zhang Z (2021) The multiple roles of ascorbate in the abiotic stress response of plants: Antioxidant, cofactor, and regulator. Front. Plant Sci 12:598173. 10.3389/fpls.2021.598173 Xu J, Shen Y, Zheng Y et al (2021) Duckweed (Lemnaceae) for potentially nutritious human food: A review. Food Reviews Int 39(7):3620–3634. 10.1080/87559129.2021.2012800 Yao XC, Meng LF, Zhao WL, Mao GL (2023) Changes in the morphology traits, anatomical structure of the leaves and transcriptome in Lycium barbarum L. under salt stress. Front Plant Sci 14:1090366. https://doi.org/10.3389/fpls.2023.1090366 Yusuf RM, Finaldin MA et al (2025) Utilization of duckweed in phytomining of gold mine tailing and its potential to produce gold nanoparticle. Environ Sci Pollut Res 32(34):20649–20658. https://doi.org/10.1007/s11356-025-36881-8 Zhang H, Xu Z et al (2020) Toxic effects of heavy metal Cd and Zn on chlorophyll, carotenoid metabolism and photosynthetic function in tobacco leaves revealed by physiological and proteomics analysis. Ecotoxicol Environ Saf 202:110856. https://doi.org/10.1016/j.ecoenv.2020.110856 Zhou W, Xin J, Tian R (2022) Photosynthetic response, antioxidase activity, and cadmium uptake and translocation in Monochoria korsakowii with cadmium exposure. Water Sci Technol 86:2974–2986. https://doi.org/10.2166/wst.2022.392 Ziegler P, Adelmann K, Zimmer S, Schmidt C, Appenroth KJ (2015) Relative in vitro growth rates of duckweeds (Lemnaceae)–the most rapidly growing higher plants. Plant Biol 17:33–41. https://doi.org/10.1111/plb.12184 Supplementary Files PlantandSoilSuplementarydata.rar Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 07 May, 2026 Reviewers invited by journal 07 May, 2026 Editor invited by journal 04 May, 2026 Editor assigned by journal 04 May, 2026 First submitted to journal 02 May, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9578120","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":636282675,"identity":"76311230-0b97-43e2-b39d-ab2d17988450","order_by":0,"name":"Rizki Maulana Yusuf","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Rizki","middleName":"Maulana","lastName":"Yusuf","suffix":""},{"id":636282676,"identity":"06f64848-41ea-4e2e-b198-f0e697cd55f7","order_by":1,"name":"Aurora Karina Chandra","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Aurora","middleName":"Karina","lastName":"Chandra","suffix":""},{"id":636282677,"identity":"e2eaddc7-2c2c-4a9a-944f-e128e3da1a14","order_by":2,"name":"Miftahudin Miftahudin","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Miftahudin","middleName":"","lastName":"Miftahudin","suffix":""},{"id":636282678,"identity":"15ed7f3d-e0b4-4e59-bad5-97bfffd24390","order_by":3,"name":"Dorly Dorly","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Dorly","middleName":"","lastName":"Dorly","suffix":""},{"id":636282679,"identity":"c3aee0ae-778f-43b5-a8cc-aceb4053b37c","order_by":4,"name":"Awalina Satya","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Awalina","middleName":"","lastName":"Satya","suffix":""},{"id":636282680,"identity":"eb7af4a1-7874-4250-ac8a-b13dea9797f4","order_by":5,"name":"Evi Susanti","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Evi","middleName":"","lastName":"Susanti","suffix":""},{"id":636282681,"identity":"1860902f-30cb-4740-8b93-9463842982e2","order_by":6,"name":"Hamim Hamim","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA20lEQVRIiWNgGAWjYBACAyA+wFAB4RxgbABRjA0ShLWcIVULA2MblAfRwsCAV4s5++nEQzfn2eWZ8x9gPFy4g0Gev4G58QY+LZY9uRsO525LLrackcBweOYZBsMZBxibLfA67ABYC3PiBqDJh3nbGBg3AN2J3y/n3wK1zKlP3HD+AFiLPWEtN0C2NBxO3HAgAawlkQgtQFtyjh0H+iWxAegXieQZhwn55Xzu5s85NdXAEDt8+HPhDhvb/vb2h3hDDAYSDICRwgyOEWZi1EO0EK94FIyCUTAKRhgAABhyUwxXNqUlAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-6811-515X","institution":"IPB: Institut Pertanian Bogor","correspondingAuthor":true,"prefix":"","firstName":"Hamim","middleName":"","lastName":"Hamim","suffix":""}],"badges":[],"createdAt":"2026-04-30 13:58:38","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9578120/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9578120/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":109760742,"identity":"67d8c465-f207-4ce9-be90-d951260af4cd","added_by":"auto","created_at":"2026-05-22 07:29:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":789684,"visible":true,"origin":"","legend":"\u003cp\u003eTwo species of duckweeds that were used in this experiment: (a) Landoltia punctata (b) Lemna aequinoctialis. r: root, mf: mother frond, dp: daughter plant. Scale bars: 2 mm\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9578120/v1/a7f8cc6e680b7e6d9a35a7ff.png"},{"id":109759414,"identity":"e31b0b78-1bcb-45b6-ba3e-1690bc3345d9","added_by":"auto","created_at":"2026-05-22 07:26:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":71512,"visible":true,"origin":"","legend":"\u003cp\u003eNumber of fronds of \u003cem\u003eL. punctata \u003c/em\u003e(a), \u003cem\u003eL. aequinoctialis\u003c/em\u003e (b) during 7 days of phytomining treatment. The media contained Hoagland solution treated with gold mine tailings (0, 4, 10 and 20%), and 138 ppm of Au prepared using Aqua regia. The values represent the average of 3 replicates\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9578120/v1/eff3dcaff1d96596076e0360.png"},{"id":109760603,"identity":"cc703b52-955c-4ec6-b9f3-e42b898bfaac","added_by":"auto","created_at":"2026-05-22 07:28:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":67946,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth parameter of duckweeds (\u003cem\u003eL. punctata \u003c/em\u003eand \u003cem\u003eL. aequinoctialis\u003c/em\u003e) during phytomining treatment using gold mine tailing (0, 4, 10 and 20%) and Au solution. (a) relative growth rate of duckweeds, (b) doubling time of duckweeds. The values represent the average of 3 replicates and the bars represent the standard deviations. Different letter indicates significantly different values between treatments based on Tukey HSD test (\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05)\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9578120/v1/e2c1fba6f8fd6af0866bdbbd.png"},{"id":109759914,"identity":"d01a389e-ff34-4438-9b9f-8bcb7b01dad6","added_by":"auto","created_at":"2026-05-22 07:27:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":853840,"visible":true,"origin":"","legend":"\u003cp\u003eMorphology of duckweed frond after 7 days of treatment. \u003cem\u003eL. punctata\u003c/em\u003e in 0% tailings (a), 4% tailings (b), 10% tailings (c), 20% tailings (d), Au 138 ppm media (e). \u003cem\u003eL. aequinoctialis \u003c/em\u003ein 0% tailings (f), 4% tailings (g), 10% tailings (h), 20% tailings (i), Au 138 ppm (j). Scale bars denote 10 mm\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9578120/v1/17033304deb4c44a1250345b.png"},{"id":109477969,"identity":"5c89e14f-e498-47f4-9e9d-2f62ee658704","added_by":"auto","created_at":"2026-05-18 14:34:33","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":35264,"visible":true,"origin":"","legend":"\u003cp\u003eMother frond area of duckweeds after 7 days of treatment using gold mine tailings (0, 4, 10 and 20%) and Au solution. The values represent the average of 3 replicates and the bars represent the standard deviations. Different letter indicates significantly different values between treatments based on Tukey HSD test (\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05)\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9578120/v1/131828ae8916ca32260c3c6e.png"},{"id":109800222,"identity":"23a5947c-30d1-427c-bf4d-586065ea0d78","added_by":"auto","created_at":"2026-05-22 15:36:55","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1577189,"visible":true,"origin":"","legend":"\u003cp\u003eTransverse cross-section of duckweed frond after 7 days of treatment using gold mine tailings (0, 4, 10 and 20%) and Au solution. Notes: \u003cem\u003eL. punctata\u003c/em\u003e in 0% tailings (a), 4% tailings (b), 10% tailings (c), 20% tailings (d), Au 138 ppm (e). \u003cem\u003eL. aequinoctialis \u003c/em\u003ein 0% tailings (f), 4% tailings (g), 10% tailings (h), 20% tailings (i), Au 138 ppm (j). ad: adaxial epidermis, ms: mesophyll, ae: aerenchyma, ab: abaxial epidermis. Scale bars:50 µm\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-9578120/v1/66c7b0e2703d3958550f3c34.png"},{"id":109760751,"identity":"fc4d03b4-3931-4496-ba4a-8677dfe45e2c","added_by":"auto","created_at":"2026-05-22 07:29:05","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":100363,"visible":true,"origin":"","legend":"\u003cp\u003eAnatomical characteristics of both duckweed fronds after 7 days of treatment using gold mine tailings (0, 4, 10 and 20%) and Au solution. (a) frond thickness, (b) mesophyll tissue thickness, (c) adaxial epidermis thickness, (d) abaxial epidermis thickness, (e) aerenchyma fraction of mesophyll tissue, (f) Ames/A. The values represent the average of 3 replicates and the bars represent the standard deviations. Different letter indicates significantly different values between treatments based on Tukey HSD test (\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05)\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-9578120/v1/fa2dcbfd5abc16d1df06f422.png"},{"id":109759739,"identity":"fbe7cf6d-8eea-4f93-bc6e-a68aae92610d","added_by":"auto","created_at":"2026-05-22 07:27:37","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":138405,"visible":true,"origin":"","legend":"\u003cp\u003ePhysiological aspect of two duckweed species (\u003cem\u003eL. punctate \u003c/em\u003eand \u003cem\u003eL. aequinoctialis\u003c/em\u003e) after 7 days of treatment. (a) H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, (b) chlorophyll a, (c) chlorophyll b, (d) total carotenoid, (e) ascorbic acid, (f) reducing sugar, (g) total phenolic, (h) proline. WW: wet weight, GE: glucose equivalent, GAE: gallic acid equivalent. The values represent the average of 3 replicates and the bars represent the standard deviations. Different letter indicates significantly different values between treatments based on Tukey HSD test (\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05)\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-9578120/v1/a6779c8ccff68ffb09acd20a.png"},{"id":109761281,"identity":"5e22c9e0-8615-4b08-bcf7-8f99af57f322","added_by":"auto","created_at":"2026-05-22 07:29:45","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":61747,"visible":true,"origin":"","legend":"\u003cp\u003eGold content in duckweeds biomass after 7 days of treatment. (a) tailings treatment media, (b) Au 138 ppm media. DW: dry weight. The values represent the average of 3 replicates and the bars represent the standard deviations. Different letter indicates significantly different values between treatments based on Tukey HSD test (\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05). *: significant differences from t-test at p \u0026lt; 0.05\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-9578120/v1/d376c60457911c5abbe0af60.png"},{"id":109760110,"identity":"a39369ae-3b56-4549-9a3f-36dddbabf1a2","added_by":"auto","created_at":"2026-05-22 07:28:10","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":240564,"visible":true,"origin":"","legend":"\u003cp\u003eGold nanoparticle from duckweeds in Au 138 ppm treatment media. (a) plant extract in water solvent (left: \u003cem\u003eL. aequinoctialis \u003c/em\u003eand right: \u003cem\u003eL. punctata\u003c/em\u003e), (b) Absorption spectrum of plant extracts. Δ: absorption peak\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-9578120/v1/bac28f92b3705e25079bf8cf.png"},{"id":109907323,"identity":"655d337e-d990-46c1-928d-44e7b0c2d451","added_by":"auto","created_at":"2026-05-25 06:42:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4751089,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9578120/v1/30327325-c09a-4623-a2a2-66aae38c7824.pdf"},{"id":109759837,"identity":"b9b430d3-02b2-4b1b-8b34-48ba6b7a5a59","added_by":"auto","created_at":"2026-05-22 07:27:49","extension":"rar","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":70035549,"visible":true,"origin":"","legend":"","description":"","filename":"PlantandSoilSuplementarydata.rar","url":"https://assets-eu.researchsquare.com/files/rs-9578120/v1/e72c66e679e2bb582e8b92a9.rar"}],"financialInterests":"","formattedTitle":"\u003cp\u003eMorphophysiological Characteristics of Two Duckweed Species Grown in Gold Mine Tailings and HAuCl\u003csub\u003e4\u003c/sub\u003e for Phytomining Purpose\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eGold is a precious and useful metal with high economic value and has been increasing all the time due to high demand. Mining activity is the primary source of gold, but with the limited availability of high-grade gold ore make people search for another alternative source. Secondary gold source is residual materials containing recoverable gold (Syed \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). One such source is gold mine tailings, the waste generated by gold mining activities. Gold mine tailings has a good prospect as an alternative gold source with the fact that tailings are produce almost 2\u0026nbsp;billion metric ton annually (Oberle et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, because of low content of gold and high content of other heavy metals, recovering gold from tailings is considered as economically unprofitable with current method of extraction (Cairncross and Tadie \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Therefore, efforts to recover gold from tailings effectively, safely, and affordably remain challenging.\u003c/p\u003e \u003cp\u003ePhytomining is a method for extracting valuable metals using hyperaccumulator plants that can accumulate these valuable metals and are harvested as a bio-ore which is more environmentally friendly to process (Sheoran et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Phytomining can become a sustainable method to extract gold from tailings, because this method is considered relatively cheap, easy to apply, and safe from environmental damage. However, this method tends only to be able to accumulate gold in small quantities and need a lot of time for the extraction process (Septian et al. 2025). According to Dinh et al. (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), gold phytomining is only considered economically profitable if the gold content in the plant biomass is more than 0.06 mg kg\u003csup\u003e-1\u003c/sup\u003e dry weight. Therefore, it is necessary to search for gold hyperaccumulator plant species.\u003c/p\u003e \u003cp\u003eAnother challenge in phytomining is that growing the plants in gold mine tailings can cause them to undergo stress due to the presence of hazardous materials in mining waste, especially high content of heavy metals (Hilmi et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Septian et al. 2025). Heavy metals in growing medium can cause stress in terrestrial as well as aquatic plants, and even many aquatic protists, due to disruption of photosynthesis (Ardipeni et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Hu et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Septian et al. 2025; Szab\u0026oacute; et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), which reduce growth. In phytomining activities, the excessive stress can reduce photosynthesis and the energy supply to plants, which inhibits growth and reduces the ability to accumulate metals (Manzoor et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Taiz et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Zhou et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Changes in morphology, anatomy, and physiology may indicate stress conditions in plants, or be part of their response to increase their ability to survive during unfavorable conditions. The reduction in growth and changes in plant morphology and physiology during the phytomining process emphasize that the plants which are utilized in a phytomining process must have resistance to heavy metals so that they can absorb as much precious metal as possible before the plants are harvested.\u003c/p\u003e \u003cp\u003eDuckweeds are fast growth aquatic plants from Lemnaceae family, several species of which, are considered as a hyper accumulator for heavy metals (Sasmaz et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Seifi and Dehghani \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and have been used in phytoremediation (Muthan et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Szab\u0026oacute; et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, the experiments utilizing duckweed for phytomining purposes, especially for gold, are still rarely found. Our previous study confirmed that a duckweed species (\u003cem\u003eLemna aequinoctialis\u003c/em\u003e) was able to extract gold up to 3.8 mg kg\u003csup\u003e-1\u003c/sup\u003e in their biomass from gold mine tailings with the addition of aqua regia solution (Yusuf et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Aqua regia itself acts as gold lixiviant which can boost gold phytomining by increase the solubility of gold and make it available for plants (Anderson et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Although lixiviant can increase the ability of plants to accumulate gold, its addition usually has a negative impact on plants, because it increases the solubility of other heavy metals in the medium, thus poisoning the plants and reducing their potential to absorb more gold (Krisnayanti et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Yusuf et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Therefore, it is necessary to obtain genotypes that are resistant to high levels of heavy metals. Duckweed family have five species which have differences in size and growth rate, so they have the potential to be further studied to explore their capability as phytomining agent. The purpose of this experiment was to observe the adjustment in morphology, anatomy, and physiology of two duckweed species (\u003cem\u003eLandoltia punctata\u003c/em\u003e and \u003cem\u003eLemna aequinoctialis\u003c/em\u003e) during phytomining and their capability to extract gold from media contained gold mine tailings as well as gold solution in the form of HAuCl\u003csub\u003e4\u003c/sub\u003e.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePreparation and acclimatization of duckweeds\u003c/h2\u003e \u003cp\u003eTwo species of duckweeds were used in this experiment which obtained from local aquatic plant farmer. Based on the identification key developed by Bog et al. (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), the duckweeds species were \u003cem\u003eLandoltia punctata\u003c/em\u003e and \u003cem\u003eLemna aequinoctialis\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These duckweeds were grown and acclimatized in separate plastic tanks (28 cm x 23 cm x 9 cm) contained 2.5 L of Hoagland's full-strength solution which were aerated and placed in a greenhouse. The plants were ready for the treatment after 14 days of acclimatization. The composition of Hoagland\u0026rsquo;s full-strength solution media followed Epstein and Bloom (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) which was composed of 6 mM of KNO\u003csub\u003e3\u003c/sub\u003e; 4 mM of Ca(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026middot;4H\u003csub\u003e2\u003c/sub\u003eO; 2 mM of [NH\u003csub\u003e4\u003c/sub\u003e]H\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e; 1 mM of MgSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO; 64 \u0026micro;M of Na[Fe(DTPA)]; 50 \u0026micro;M of KCl; 25 \u0026micro;M of H\u003csub\u003e3\u003c/sub\u003eBO\u003csub\u003e3\u003c/sub\u003e; 2 \u0026micro;M of MnSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;H\u003csub\u003e2\u003c/sub\u003eO; 2 \u0026micro;M of ZnSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO; 0.5 \u0026micro;M of CuSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;5H\u003csub\u003e2\u003c/sub\u003eO; and 0.5 \u0026micro;M of H\u003csub\u003e2\u003c/sub\u003eMoO\u003csub\u003e4\u003c/sub\u003e. All nutrients were dissolved in distilled water.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eLeaching mineral of gold mine tailings\u003c/h3\u003e\n\u003cp\u003eThe preparation and leaching of gold mine tailings used in the experiment was based on the method from our previous study (Yusuf et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Gold mine tailings were obtained from tailings dam facility of Indonesian Gold Company, Aneka Tambang (ANTAM) Inc., UBPE Pongkor, Bogor, West Java, Indonesia (6\u0026deg; 38\u0026prime; 45\u0026Prime; S, 106\u0026deg; 34\u0026prime; 20\u0026Prime; E). The leaching process was performed by slowly mixing 4 kg of dry fine tailings with 2 L of aqua regia solution until it evenly mixed and forms paste-like tailings suspension and allowed to stand for 24 hours. After 24 hours, 5% dolomite suspension was used to increase the pH level of tailings suspension up to 5.0 and then allowed to stand for another 24 hours.\u003c/p\u003e\n\u003ch3\u003ePreparation of gold solution\u003c/h3\u003e\n\u003cp\u003eThe gold(III) chloride (HAuCl\u003csub\u003e4\u003c/sub\u003e) stock solution with a concentration of 0.5 \u003cem\u003eM\u003c/em\u003e was prepared by dissolving 2 g of pure gold in 18.2 mL aqua regia solution following the procedure made by Mayoral et al. (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The mixture was left for 48 hours for residual acids removal, then mixed in Hoagland\u0026rsquo;s media solution and used as a positive control to observe the ability of duckweed to accumulate pure gold ion from the culture media.\u003c/p\u003e\n\u003ch3\u003eGold phytomining process by duckweeds\u003c/h3\u003e\n\u003cp\u003eAfter 14 days of acclimatization in Hoagland\u0026rsquo;s media, the duckweeds then were grown in the media contained gold mine tailings or gold solution for phytomining experiment. The experiment was carried out following the method developed previously by Yusuf et al. (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) with some modification. The experiment was prepared using Completely Randomized Design with three experimental repetition and two variables: duckweed species (\u003cem\u003eL. punctata\u003c/em\u003e and \u003cem\u003eL. aequinoctialis\u003c/em\u003e) and treatment media. There were five media treatments that used in this experiment. The first, second, third, dan fourth treatment were tailings treatment media with the concentration of 0% (negative control), 4%, 10%, and 20% of tailings suspended to Hoagland\u0026rsquo;s full-strength solution. The tailings treatment medium was prepared by mixing 0, 100, 250, and 500 g of tailings suspension with Hoagland\u0026rsquo;s media until it reached a volume of 2.5 L. The fifth treatment was Hoagland\u0026rsquo;s full-strength solution with the addition of HAuCl\u003csub\u003e4\u003c/sub\u003e solution at 0.7 mM labeled as Au 138 ppm medium that was used as positive control to study the responses of duckweeds to gold (Au) ion. Au 138 ppm medium was prepared by diluting 3.26 mL of HAuCl\u003csub\u003e4\u003c/sub\u003e stock solution with Hoagland\u0026rsquo;s full-strength solution until it reached a volume of 2.5 L. Before duckweed planting, all the treatment were aerated for 48 hours and then the pH of all treatment was adjusted to became 6.0 using dolomite. A 50 g of acclimatized duckweeds were weighed and then were grown and maintained into each treatment medium for seven days. All treatment media were plastic tanks (28 cm x 23 cm x 9 cm) equipped with a plastic cup (with diameter 5.5 cm and height 9 cm) that was glued on plastic tank for specific measurement. The plastic cup was perforated for circulation of media between the tank and the cup. Four duckweed plants with similar size and number frond were placed in the plastic cup that was already glued in the plastic tank for monitoring their growth and morphology changes.\u003c/p\u003e\n\u003ch3\u003eGrowth and morphology monitoring\u003c/h3\u003e\n\u003cp\u003eThe frond number of duckweeds grown in the plastic cup placed on each treatment was counted every day for 7 days to calculate the growth. Relative growth rate (RGR) and doubling time (DT) per day were calculated from the number of fronds (NF) in day 1 and day 7 using Eqs.\u0026nbsp;(\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and (\u003cspan refid=\"Equ2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) respectively (Ziegler et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). After 7 days of treatment, the picture of duckweed frond on each treatment media was taken using a camera with the same distance to monitor morphological changes. The pictures were then given a scale bar using ImageJ and the mother frond area of each duckweed plant was calculated using freehand tool of ImageJ with two repeated measurements.\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:RGR=\\frac{\\text{ln}NF\\:day\\:7-\\text{ln}NF\\:day\\:1}{day\\:7-\\:day\\:1}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e\n$$\\:DT=\\frac{\\text{ln}2\\:}{RGR}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eHarvesting and preparation of duckweed samples\u003c/h2\u003e \u003cp\u003eAfter 7 days planting, duckweeds were harvested, cleaned with running distilled water and then drained using paper towel. After dry, the duckweed was weighed to measure the harvested wet weight. For physiological test samples, 10 g of fresh duckweed from each treatment was drenched in liquid nitrogen and keep in a cooling box before the samples were stored in -80\u0026deg;C freezer to keep them fresh. The duckweed from plastic cup was also cleaned and then was fixed for anatomy observation using Formaldehyde Alcohol Acetic Acid (FAA) solution for 24 hours. After 24 hours, the sample then was drenched three times in 50% ethanol solution for 15 mins each and then was stored in 70% ethanol solution. The rest of the sample which was not used for anatomy or physiology analysis will be used for the measurement of water content and gold content analysis.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAnatomy observation of duckweed\u003c/h3\u003e\n\u003cp\u003eTransverse cross-section of duckweed frond was prepared using paraffin embedded cross-section method (Johansen \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1940\u003c/span\u003e). After it was stored in 70% ethanol solution, the fronds then were dehydrated using a series of Johansen solutions I-VII as dehydrants (Johansen \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1940\u003c/span\u003e). After that, dehydrated fronds were embedded in paraffin block and then were drenched in Gifford solution to soften the sample (Gifford \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1950\u003c/span\u003e). The embedded samples were then sliced transversally using rotary microtome (Yamato RV-240) with thickness of 10 \u0026micro;m. The paraffin from the frond cross-section then washed using xylene and then the cross-section was observed using bright field microscope (Olympus Cx23 with Indomicro camera). The picture of transversal cross-section was captured from the left side and the right side of midrib of frond with three replications for each treatment. The pictures then were analyzed using ImageJ with two repeated measurement which the parameters that were analyzed including frond thickness, epidermis thickness (adaxial and abaxial), mesophyll tissue thickness (MT), mesophyll tissue area, aerenchyma area, and mesophyll cells perimeter (representative) (Pmc), and number of mesophyll cells (Nmc). Aerenchyma fraction of mesophyll tissue (AFM) and the surface area of mesophyll cells per leaf area (Ames/A) was calculated using equations (\u003cspan refid=\"Equ3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) and (\u003cspan refid=\"Equ4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) from Sack et al. (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) respectively with modification which in the Ames/A calculation, Pmc was based on the average of 10 representative cells which chosen randomly. There were 2 group of representative cells which were small cells group (Pmc\u0026thinsp;\u0026lt;\u0026thinsp;100 \u0026micro;m) and large cells group (Pmc\u0026thinsp;\u0026ge;\u0026thinsp;100 \u0026micro;m). Mesophyll cells were modeled as spheres for estimated mesophyll cell surface area (SAmc) and mesophyll cell volume (Vmc) (Eqs.\u0026nbsp;\u003cspan refid=\"Equ5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and \u003cspan refid=\"Equ6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003cdiv id=\"Equ3\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ3\" name=\"EquationSource\"\u003e\n$$\\:AFM=\\:\\frac{\\sum\\:Arenchyma\\:tissue\\:area}{Mesophyll\\:tissue\\:area\\:}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e3\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equ4\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ4\" name=\"EquationSource\"\u003e\n$$\\:\\text{A}\\text{m}\\text{e}\\text{s}/\\text{A}=\\:\\frac{\\sum\\:SAmc\\times\\:\\left(MT\\left(1-AFM\\right)\\right)}{\\sum\\:Vmc\\:}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e4\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equ5\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ5\" name=\"EquationSource\"\u003e\n$$\\:SAmc=\\left(4\\pi\\:\\times\\:{\\left(\\frac{Pmc}{2\\pi\\:}\\right)}^{2}\\right)\\times\\:Nmc$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e5\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equ6\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ6\" name=\"EquationSource\"\u003e\n$$\\:Vmc=\\left(\\frac{4}{3}\\pi\\:\\:\\times\\:{\\left(\\frac{Pmc}{2\\pi\\:}\\right)}^{3}\\right)\\:\\times\\:Nmc$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e6\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003ePhysiological analysis of duckweed\u003c/h3\u003e\n\u003cp\u003ePhysiological aspect of duckweed which were analyzed including H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content, photosynthetic pigment content, ascorbic acid content, total phenolic content, reducing sugar content, and proline content. All the samples were in fresh condition and crushed with addition of liquid nitrogen. H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content was measured using a method from Junglee et al. (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) with KI reagent and then the absorbance of sample extract was measured using a UV-vis spectrophotometer (Metertech SP-8001) in 285 nm wavelength. Photosynthetic pigment content which consisted of chlorophyll a, chlorophyll b, and total carotenoids were measured using method from previous study (Wellburn \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e1994\u003c/span\u003e) which the pigment was extracted using 80% acetone solution and then the absorbance of extract was measured in wavelengths of 470, 646, and 663 nm. The equations from the same studies were used to calculate the content of chlorophyll a, chlorophyll b, and total carotenoids which can be seen in Eq.\u0026nbsp;\u003cspan refid=\"Equ7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, \u003cspan refid=\"Equ8\" class=\"InternalRef\"\u003e8\u003c/span\u003e, \u003cspan refid=\"Equ9\" class=\"InternalRef\"\u003e9\u003c/span\u003e respectively. Ascorbic acid from the plant was measured using Abera et al. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) method which the plant extract was reacted with 0.335 mM K\u003csub\u003e2\u003c/sub\u003eCr\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e and 0.185 mM MnCl\u003csub\u003e2\u003c/sub\u003e and then the absorbance was measured twice immediately at 350 nm and later after 5 mins delay. The method from Ghanem et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) was used to measure the total phenolic content which the plant extract was reacted with Folin Ciocalteu reagent and then the absorbance was measured at 765 nm. Reducing sugar content from duckweed was measured using a method from Khatri and Chhetri (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) which hot 3,5-Dinitrosalicylic acid reagent was reacted with plant extract and then the absorbance was measured at 540 nm. Proline content was measured using method from Schweet (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1954\u003c/span\u003e) which the plant extract was reacted with Acid Ninhydrin reagent to produce chromogen then was extracted using toluene and the absorbance of chromogen was measured at 520 nm. All the physiological measurement was done twice.\u003cdiv id=\"Equ7\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ7\" name=\"EquationSource\"\u003e\n$$\\:Chlorophyll\\:a=12.25\\left(Absorbance\\:663\\:nm\\right)-2.79\\left(Absorbance\\:646\\:nm\\right)$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e7\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equ8\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ8\" name=\"EquationSource\"\u003e\n$$\\:Chlorophyll\\:b\\:=21.5\\left(Absorbance\\:646\\:nm\\right)-2.79\\left(Absorbance\\:663\\:nm\\right)$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e8\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equ9\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ9\" name=\"EquationSource\"\u003e\n$$\\:Total\\:carotenoid=\\frac{1000\\left(Absorbance\\:470\\:nm\\right)-1.82\\left(Chlorophyll\\:a\\right)-85.02\\left(Chlorophyll\\:b\\right)}{198}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e9\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePlant dry weight measurement\u003c/h2\u003e \u003cp\u003eThe fresh weight of sample from each treatment was measured then the sample was dried in oven at 60\u0026deg;C for 72 hours. After dried, the sample weight was measured again as dry weight. The water content of duckweed was measured base on the changes of plant weight before and after drying process (Eq.\u0026nbsp;\u003cspan refid=\"Equ10\" class=\"InternalRef\"\u003e10\u003c/span\u003e). Harvest dry weight of duckweed from each treatment media was estimate based on harvest wet weight and water content (Eq.\u0026nbsp;\u003cspan refid=\"Equ11\" class=\"InternalRef\"\u003e11\u003c/span\u003e).\u003cdiv id=\"Equ10\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ10\" name=\"EquationSource\"\u003e\n$$\\:Water\\:content=\\frac{\\left(wet\\:weight-dry\\:weight\\right)}{wet\\:weight}\\:\\times\\:100\\%$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e10\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equ11\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ11\" name=\"EquationSource\"\u003e\n$$\\:Harvested\\:biomass=harvested\\:wet\\:weight\\:\\times\\:\\left(100\\%-water\\:content\\right)$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e11\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eGold content analysis and phytomining aspect analysis\u003c/h2\u003e \u003cp\u003eThe gold content analysis in duckweed biomass and tailings followed the method from Satya et al. (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) with modification to adjust with gold sample. The analysis was started with the destruction of 0.1 g of dry sample in digestion vessel with mixed solution of 3 mL of 30% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, 3 mL of concentrated HNO\u003csub\u003e3\u003c/sub\u003e, and 5 mL of deionized water then the mixture was heated using an autoclave at a temperature of 121\u0026deg;C with a pressure of 1 atm for 45 mins. After heated, the pH of mixture was raised to 2 by adding 1 N KOH solution and then 2 mL of 6 M KSCN solution was added into the mixture and allowed to stand for 24 hours to dissolve the remaining gold. After 24 hours, the mixture was added again with 1 mL of 30% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and 1 mL of concentrated HNO\u003csub\u003e3\u003c/sub\u003e and then was heated again in the autoclave with the same parameter as before to obtain a clear digestate. The gold content from digestate was then measured using a graphite furnace atomic absorption spectrophotometer (GF-AAS) (Hitachi Z 2700). The potential of duckweed used as gold phytomining agent was looked from bio-concentration factor (BCF) Bioconcentration factor was calculated following the equation from Verma \u0026amp; Suthar (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) which a ratio between gold content in plant with gold concentration in media (Eq.\u0026nbsp;\u003cspan refid=\"Equ12\" class=\"InternalRef\"\u003e12\u003c/span\u003e). Gold concentration for tailings treatment media was estimated from the amount of tailings in the media and the tailings gold content.\u003cdiv id=\"Equ12\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ12\" name=\"EquationSource\"\u003e\n$$\\:BCF=\\frac{Gold\\:content\\:in\\:biomass\\:}{Initial\\:gold\\:concentration\\:in\\:media}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e12\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe statistical test two-way ANOVA with \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was used in this research unless it was stated using different statistical test. Significantly different tests resulted from ANOVA were then analyzed further using Tukey HSD analysis. The statistical analyses were done in IBM SPSS Statistic 25.\u003c/p\u003e \u003c/div\u003e "},{"header":"RESULTS","content":"\u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003eGold mine tailings and gold solution decreased the growth of duckweeds\u003c/h2\u003e \u003cp\u003eThe growth of duckweed measured based on frond number, relative growth rate and doubling growth was influenced strongly by gold mine tailings as well as gold solution. The changes of fronds number in both duckweed species during 7 days treatment can be seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. In media without addition of tailings or gold solution (0% media), the growth of frond number was faster on L. aequinoctialis compared to L. punctata. The ability of L. aequinoctialis to grow faster compared to L. punctata also can be seen in media with high tailings (20%) or gold content (Au 138 ppm) which there was a slight growth on number of fronds each day for L. aequinoctialis while the number of fronds of L. punctata did not change after day 2 until day 7 of treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Other growth parameters (relative growth rate and doubling time) also had similar trend (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The relative growth rate of L. aequinoctialis was significantly higher than L. punctata in 0% tailings media. The relative growth rate of these two plants was decline along with the increase of tailing content in media. The 138 ppm gold in Hoagland\u0026rsquo;s media (Au 138 ppm) also caused a significant decrease on relative growth rate in both plants. Lastly, doubling time for L. aequinoctialis did not show a significant difference between each treatment while L. punctata in 20% tailing and Au 138 ppm media had significantly longer doubling time compared to negative control media.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eMorphological responses of duckweed during gold phytomining\u003c/h2\u003e \u003cp\u003eDuring 7 days of treatment using 4, 10 and 20% of gold mine tailing and Au solution, the plants underwent morphological changes as compared to control plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The color of \u003cem\u003eL. punctata\u003c/em\u003e fronds after 7 days treatment in media that consisted of 0%, 4%, 10%, and 20% shifted from green into pale-yellow (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, b, c, and d). The same changes on fronds color between tailing treatment media also happened to \u003cem\u003eL. aequinoctialis\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef, g, h, and i). In contrast, the frond color of these two species was turning into purple after grew in Au 138 ppm media for 7 days. The area of mother fronds of \u003cem\u003eL. punctata\u003c/em\u003e and \u003cem\u003eL. aequinoctialis\u003c/em\u003e were not significantly different for all treatments media for each duckweed species (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eFrond anatomy changes of duckweeds during gold phytomining\u003c/h2\u003e \u003cp\u003eThe transverse cross-section of frond from both duckweed species suggested that gold mine tailings and Au solution treatments reduced the thickness of frond anatomy on both species, especially due to the reduction of mesophyll cells due to the treatments, except on \u003cem\u003eL. punctata\u003c/em\u003e grown at 10% of gold mine tailing (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Frond thickness of \u003cem\u003eL. punctata\u003c/em\u003e in 10% tailings treatment media was the highest compared to other treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea) and it can be associated with their mesophyll tissue thickness which had the same pattern (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb). However, for \u003cem\u003eL. aequinoctialis\u003c/em\u003e which grew in tailings treatment media, the thickness of frond and mesophyll were decreasing with the increase of tailing content in media. The thickness of adaxial epidermis from \u003cem\u003eL. aequinoctialis\u003c/em\u003e did not have a significant difference between all treatment media while for \u003cem\u003eL. punctata\u003c/em\u003e which grew in 10 and 20% tailings media had significantly thicker adaxial epidermis compared to 0% tailings media (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec). The cross-section of these two duckweeds frond which grew in Au 138 ppm media showed that this treatment caused the thickness of frond and mesophyll tissue thinner compared to their respective negative control (0% tailings treatment). Furthermore, this treatment also caused the cells of these duckweed fronds turning color into purple with the lower part of frond (abaxial) had darker coloration compared to upper part (adaxial).\u003c/p\u003e \u003cp\u003eFor abaxial epidermis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed), \u003cem\u003eL. punctata\u003c/em\u003e that grew in 20% tailings treatment media had thicker abaxial epidermis compared with other treatment while for \u003cem\u003eL. aequinoctialis\u003c/em\u003e, the plant that had the thickest abaxial epidermis was \u003cem\u003eL. aequinoctialis\u003c/em\u003e in 0% tailing treatment media. The fraction of aerenchyma in mesophyll tissue of duckweeds frond can be seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee. The fraction of aerenchyma of \u003cem\u003eL. punctata\u003c/em\u003e in all treatment media did not have significant different from each other. For \u003cem\u003eL. aequinoctialis\u003c/em\u003e, the plant that grew in 10% tailing treatment media had significantly large total area of aerenchyma in their mesophyll tissue compared to other treatment. for Ames/A of \u003cem\u003eL. punctata\u003c/em\u003e, all plant in tailings treatment media did not have significant different while Ames/A for \u003cem\u003eL. punctata\u003c/em\u003e in Au 138 ppm treatment was significantly lower compared to negative control (0% tailings treatment) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee). The Ames/A of \u003cem\u003eL. aequinoctialis\u003c/em\u003e in 0% and 4% tailings treatment media were higher compared to other three treatments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003ePhysiological responses of duckweed during phytomining\u003c/h2\u003e \u003cp\u003eTo understand the physiological responses of the duckweeds, some parameters including the content of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, photosynthetic pigments, ascorbic acid, reducing sugar, phenolic as well as proline were measured. H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels in duckweeds after 7 days of treatment can be seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea. It can be seen that \u003cem\u003eL. aequinoctialis\u003c/em\u003e showed an increase in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels in plant grown on media containing tailings (4%, 10%, 20% tailings media) when compared to control negative (0%). Meanwhile, \u003cem\u003eL. punctata\u003c/em\u003e did not show a clear pattern with the 4% and 10% tailings treatments, causing a decrease in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels, but the 20% tailings treatment was not significantly different from the control. Both duckweed species that grown in the 138 ppm Au treatment showed relatively fewer H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels than the control. The contents of chlorophyll a, chlorophyll b, and total carotenoids can be seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb, c, and d respectively. The content of these three photosynthetic pigments is significantly reduced in both of plants grown in media containing 20% tailings. The response of the two duckweeds after being grown in media containing 138 ppm gold (Au 138 ppm) for seven days showed a decrease in the levels of photosynthetic pigments compared to the control (0% tailings treatment).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAscorbic acid levels in duckweeds after seven days of treatment can be seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ee. \u003cem\u003eL. punctata\u003c/em\u003e grown in 10%, 20% tailings, and Au 138 ppm treatment media showed ascorbic acid levels that were significantly higher than the control. \u003cem\u003eL. aequinoctialis\u003c/em\u003e grown in 4% tailings media showed lower ascorbate levels than the control. The reducing sugar levels of duckweeds grown for seven days can be seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ef. The reducing sugar levels in \u003cem\u003eL. punctata\u003c/em\u003e and \u003cem\u003eL. aequinoctialis\u003c/em\u003e in the 10% tailings treatment showed the highest levels compared to other tailings treatments. In the 138 ppm Au treatment, both plants showed a decrease in reducing sugar levels compared to the control. Figure\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eg shows duckweeds total phenolic content after seven days of treatment. Total phenolic content of \u003cem\u003eL. punctata\u003c/em\u003e showed a significant decrease as the level of tailings added to the media increased. Meanwhile, for \u003cem\u003eL. aequinoctialis\u003c/em\u003e, the total phenolic content in each tailings treatment did not show significant differences. Both of plants which grew in 138 ppm Au treatment showed significantly lower levels of total phenolics and flavonoids than the control. Figure\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eh shows the proline levels in duckweeds after seven days of treatment. The proline levels of \u003cem\u003eL. punctata\u003c/em\u003e grown in 20% tailings media showed significantly higher proline levels than other treatments. Meanwhile, the proline levels of \u003cem\u003eL. aequinoctialis\u003c/em\u003e in each treatment did not show significant differences.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eGold accumulation and gold nanoparticles synthesis in duckweeds\u003c/h2\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea shows the accumulation of gold in duckweed biomass after seven days of treatment in tailings. The gold content in plant that grew on 0% tailings media is considered as the lowest gold concentration that generally found in the duckweed (basal content). In 4% tailings treatment media, \u003cem\u003eL. punctata\u003c/em\u003e did not show a significant gold accumulation compared to the 0% tailings media treatment while the gold content of \u003cem\u003eL. aequinoctialis\u003c/em\u003e in 4% tailings treatment media was significantly higher than their 0% treatment media. Gold accumulation in both plants significantly increased compared to the 0% tailings media treatment when the plants were grown in the 10% tailings media treatment. However, adding 20% tailings suspension to the media did not cause a more significant increase in gold accumulation compared to the 10% tailings treatment in both plants.\u003c/p\u003e \u003cp\u003eIn the 138 ppm Au treatment, it is known that gold accumulation by \u003cem\u003eL. punctata\u003c/em\u003e and \u003cem\u003eL. aequinoctialis\u003c/em\u003e can reach 2% of plant biomass (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eb). Gold content in this treatment is much higher than that of plants grown in all tailings media treatment. \u003cem\u003eL. punctata\u003c/em\u003e on 138 ppm Au treatment media can accumulate gold up to 23.3 mg g\u003csup\u003e-1\u003c/sup\u003e on average, which is significantly higher than gold accumulation on \u003cem\u003eL. aequinoctialis\u003c/em\u003e (18.0 mg g\u003csup\u003e-1\u003c/sup\u003e). The plants that grew in Au 138 ppm media were extracted of their gold nanoparticles because of high gold accumulation. The color of extract from \u003cem\u003eL. punctata\u003c/em\u003e was dark purple while for \u003cem\u003eL. aequinoctialis\u003c/em\u003e had purple coloration (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003ea). Based on absorption spectrum of extracts, it is known that both showed LSPR for spherical gold nanoparticle (peak at wavelength 500\u0026ndash;550 nm) with the \u003cem\u003eL. aequinoctialis\u003c/em\u003e extract have absorption peak at 538 nm while \u003cem\u003eL. punctata\u003c/em\u003e had absorption peak at 545 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003ePhytomining aspects of duckweeds grown in 4, 10, and 20% tailings treatment media can be seen in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. \u003cem\u003eL. aequinoctialis\u003c/em\u003e has a relatively higher harvested wet weight than \u003cem\u003eL. punctata\u003c/em\u003e. However, \u003cem\u003eL. punctata\u003c/em\u003e has a relatively higher harvested dry weight than \u003cem\u003eL. aequinoctialis\u003c/em\u003e. The water content in \u003cem\u003eL. punctata\u003c/em\u003e was relatively lower compared to \u003cem\u003eL. aequinoctialis.\u003c/em\u003e Tailings sample used in this study had a gold content of 4.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.458 \u0026micro;g g\u003csup\u003e-1\u003c/sup\u003e, and the gold concentration in tailings treatment media was even smaller due to the dissolution process. The bioconcentration factor (BCF) value of both duckweeds showed a high value (BCF\u0026thinsp;\u0026gt;\u0026thinsp;1). Based on mass gold in plant, it is known that L. punctata grown in 10% tailings treatment media can produce gold that is higher than other treatments. Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the phytomining aspects of the two duckweeds species grown in 138 ppm Au treatment media which containing gold solution (HAuCl\u003csub\u003e4\u003c/sub\u003e). In this treatment, the wet and dry harvested weight did not show significant differences between the two duckweed species. The water content of the two plants is also similar. The bioconcentration factors of Au from \u003cem\u003eL. punctata\u003c/em\u003e and \u003cem\u003eL. aequinoctialis\u003c/em\u003e were 179 and 144, respectively. The mass of gold that was successfully harvested from both plants in this medium was much higher than the tailings treatment, with the average of mass gold in plant from \u003cem\u003eL. punctata\u003c/em\u003e being higher than \u003cem\u003eL. aequinoctialis\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAspects of the gold phytomining process in gold mine tailings by duckweeds\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAspect\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"7\" nameend=\"c9\" namest=\"c3\"\u003e \u003cp\u003eDuckweed species\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003e\u003cem\u003eL. punctata\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c9\" namest=\"c7\"\u003e \u003cp\u003e\u003cem\u003eL. aequinoctialis\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTailing content in the media\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e20%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e20%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGold concentration in media (mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003csup\u003eΔ\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGold content in biomass (\u0026micro;g g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBCF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHarvested wet weight (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29.6\u0026thinsp;\u0026plusmn;\u0026thinsp;4.35\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e34.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.34\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e39.8\u0026thinsp;\u0026plusmn;\u0026thinsp;6.84\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e46.9\u0026thinsp;\u0026plusmn;\u0026thinsp;5.04\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e49.6\u0026thinsp;\u0026plusmn;\u0026thinsp;10.18\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e42.7\u0026thinsp;\u0026plusmn;\u0026thinsp;10.26\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWater content (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e90\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e91\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e95\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e95\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHarvested biomass (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMass gold in plant (\u0026micro;g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.76\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003eΔ\u003c/sup\u003e: Media gold content is estimated from the amount of tailings in the media where the gold content in the tailings was 4.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.458 \u0026micro;g g\u003csup\u003e-1\u003c/sup\u003e. BCF: bioconcentration factor. The values represent the average of 3 replicates. Different superscript letters indicate significant differences from Tukey HSD test at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. \u0026plusmn;: standard deviation\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAspects of the gold phytomining process in gold solution (HAuCl\u003csub\u003e4\u003c/sub\u003e) by duckweeds\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAspect\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003eDuckweed species\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eL. punctata\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eL. aequinoctialis\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAu content in the media\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAu 138 ppm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAu 138 ppm\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGold content in media (mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e138\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e138\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGold content in biomass (mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.36\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.95\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBCF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e169\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e131\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHarvested wet weight (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e40.2\u0026thinsp;\u0026plusmn;\u0026thinsp;15.05\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e35.5\u0026thinsp;\u0026plusmn;\u0026thinsp;6.28\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWater content (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHarvested biomass (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.86\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMass gold in plant (mg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e53.5\u0026thinsp;\u0026plusmn;\u0026thinsp;20.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e33.5\u0026thinsp;\u0026plusmn;\u0026thinsp;5.92\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eBCF: bioconcentration factor. The values represent the average of 3 replicates. Different superscript letters indicate significant differences from Tukey HSD test at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. \u0026plusmn;: standard deviation\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eGrowth and morpho-physiological characteristics of duckweed in phytomining process\u003c/h2\u003e \u003cp\u003eDuckweed is an aquatic plant belongs to Lemnaceae family (Xu et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) which has very rapid growth rates. In this study, the two duckweeds had different growth rates, i.e.: 3.1 fronds per day for \u003cem\u003eL. punctata\u003c/em\u003e and 7.3 fronds per day for \u003cem\u003eL. aequinoctialis\u003c/em\u003e in the negative control treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). However, both species experienced a significant decrease in growth rate when treated with gold mine tailings and Au solution, and the rate of decline was consistent with the increase in tailings concentration (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This indicates that the tailings and Au solution treatments negatively impacted both duckweeds. Based on previous studies by Hilmi et al. (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) showed that gold mine tailings collected from an Indonesian gold company, Aneka Tambang Inc. contained several metal and heavy metals with high concentrations including magnesium (Mg): 3962.71 ppm, iron (Fe): 10348.15 ppm and Manganese (Mn): 1791.46 ppm, lead (Pb): 93 ppm, silver (Ag): 13 ppm, and cadmium (Cd): 1 ppm. The use of aqua regia compounds as gold lixiviant in tailings caused an increase in the solubility of heavy metals contained in the tailings (Yusuf et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) so that they were easily absorbed by plants and consequently were toxic to the duckweeds within 7 days of treatment. Cao et al. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) also reported that \u003cem\u003eLemna minor\u003c/em\u003e and \u003cem\u003eL. punctata\u003c/em\u003e had similar growth rates when they grew in control media and then it declined significantly with almost the same growth rate when these two duckweeds were grown in 10 mg L\u003csup\u003e-1\u003c/sup\u003e of cadmium (Cd) media. Apart from tailings, the study also revealed that Au\u003csup\u003e3+\u003c/sup\u003e solution in the form of HAuCl\u003csub\u003e4\u003c/sub\u003e also significantly hindered the duckweeds growth (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This data is also in accordance with Taylor et al. (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) who revealed that K(AuCl\u003csub\u003e4\u003c/sub\u003e) in concentration of 200 mg L\u003csup\u003e-1\u003c/sup\u003e significantly inhibited the growth of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e root.\u003c/p\u003e \u003cp\u003eIn addition to growth, the level of stress on duckweed due to gold mine tailings and gold solution treatment can also be observed in the change of frond colour of both duckweeds which changed from green to whitish yellow (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The colour change was verified by the decrease in the content of photosynthetic pigments, including chlorophyll \u003cem\u003ea\u003c/em\u003e, chlorophyll \u003cem\u003eb\u003c/em\u003e and carotenoids (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb, c, and d). This decrease in plant pigments was probably due to heavy metal stress from tailings and HAuCl\u003csub\u003e4\u003c/sub\u003e solution treatment. Several studies have also shown that plants experiencing heavy metal stress underwent a decrease in chlorophyll content (Li et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Heavy metal treatment can stimulate oxidative stress due to the formation of large amounts of reactive oxygen species (ROS) such as hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), superoxide (O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e), and hydroxyl radicals (HO\u003csup\u003e\u0026bull;\u003c/sup\u003e) (Hu and Wang \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Sood \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this experiment, only \u003cem\u003eL. aequinoctialis\u003c/em\u003e experienced an increase in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in response to the tailings treatment, while \u003cem\u003eL. punctata\u003c/em\u003e did not (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea). This is suspected to be related to the \u003cem\u003eL. punctata\u003c/em\u003e genotype's ability to control ROS through a good antioxidant system, as indicated by the increase in ascorbic acid and proline content in \u003cem\u003eL. punctata\u003c/em\u003e, especially in the tailings treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ee and h). There are 2 hypothesis that could explain the reduced H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content in Au 138 ppm treatment compared to negative control (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea). The first hypothesis is H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content had peaked earlier than the sampling time which it could happen when the plant activated ROS scavenging mechanism after sensing the increased H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content in cells. Kabala et al. (2022) reported that treatment with high concentration of NaCl stimulated the formation of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, in \u003cem\u003eCucumis sativus\u003c/em\u003e on the first day of treatment, although it decreased again after 6 days of stress. This decrease can be corelated with their catalase activity which increased significantly in 6 days of treatment (Kabala et al. 2022). The second hypothesis is gold nanoparticles in plant cells can catalyzed the decomposition of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e into hydroxyl radicals (HO\u003csup\u003e\u0026bull;\u003c/sup\u003e) or O\u003csub\u003e2\u003c/sub\u003e and H\u003csub\u003e2\u003c/sub\u003eO depends on pH (He et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAscorbic acid, carotenes, tocopherols, and glutathione are commonly compounds produced by plants in response to environmental stressors (Gupta et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Ascorbic acid is an important antioxidant for plants in controlling ROS levels (Xiao et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The reaction between ascorbic acid and free radicals like ROS produces dehydroascorbic acid, which is an oxidized form of ascorbic acid. Jung et al. (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) showed that Oryza sativa seedlings grown in media with arsenic (As) did not increase their ascorbic acid, but their ROS scavenging activity may be correlated with the increase in dehydroascorbic acid. In addition, proline, besides has been known as an osmolyte compound in the face of osmotic stress, also plays an important role in controlling ROS in tissues (Renzetti et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In some species, a slight increase in ROS concentration due to mild stress can act as oxidative signalling, which temporarily increases the ROS scavengers to balance the ROS accumulation (Sood \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The high ROS and low ascorbate levels in \u003cem\u003eL. aequinoctialis\u003c/em\u003e during tailing treatment may indicate a relatively lower resistance level of this species to tailing treatment and may be an indication of why the photosynthetic pigment content in this species was significantly lower than that of \u003cem\u003eL. punctata\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea and e). Another common antioxidant in plants is phenolic which often increases when plants experience stress condition. However, under tailings treatment, the phenolic content of \u003cem\u003eL. punctata\u003c/em\u003e decreased as the content of tailings in media increase while \u003cem\u003eL. aequinoctialis\u003c/em\u003e did not show any significant change in their phenolic content in all tailings treatment media (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eg. Under certain circumstances, severe salinity stress decreased the production of phenolic compound by downregulating phenylalanine ammonia-lyase (PAL) enzyme which involved in phenolic biosynthesis in plants (Mr\u0026aacute;zov\u0026aacute; et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In addition, studies from Pungin et al. (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) revealed that adding NaCl up to 100 mM can increase phenolic content of \u003cem\u003eGlaux maritim\u003c/em\u003e plant, but then the phenolic content drop when the concentration of NaCl exceeded 100 mM. The reason behind the phenolic content decreasing during severe salinity stress is still unknown but it is believed that plants focus to produce small molecule osmolyte such as proline and simple sugar when the cells osmotic pressure was high.\u003c/p\u003e \u003cp\u003eThe results of frond anatomy analysis also showed that treatment with tailings and gold solution caused a decrease in frond thickness, especially due to a significant decrease in mesophyll cell thickness in both duckweed species, except for \u003cem\u003eL. punctata\u003c/em\u003e in the 10% tailings treatment which experienced an increase (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea and b). The decrease in frond thickness is suspected to be due to ultrastructural damage of frond cells due to the effects of heavy metals, as observed by Baruah et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) in the Cd treatment, and Prasetya et al. (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) in \u003cem\u003eJ. curcas\u003c/em\u003e and \u003cem\u003eR. trisperma\u003c/em\u003e grown in media containing 100% gold mine tailings. Rucińska-Sobkowiak (2016) also mentioned that the anatomical change in plants caused by heavy metals can be directly from heavy metal toxicity in plant cells or indirectly from abscisic acid (ABA) stress signaling in plants. The increase of mesophyll cells in \u003cem\u003eL. punctata\u003c/em\u003e is suspected to be related to hormesis response. Hormesis response is phenomenon where low intensity stressor has beneficial effect for plants (Agathokleous et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). These responses including the increased of frond thickness, mesophyll thickness, ascorbic acid, and reducing sugar showed the level of resistance of this species to stress. Decreased leaf thickness is a common symptom experienced by plants exposed to heavy metal stress. Using 4 species of non-edible oil-producing plants (\u003cem\u003eJatropha curcas\u003c/em\u003e, \u003cem\u003eRicinus communis\u003c/em\u003e, \u003cem\u003eMelia azedarach\u003c/em\u003e and \u003cem\u003eReutealis trisperma\u003c/em\u003e) Hamim et al. (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) found that the treatment using mercury in the form of Hg(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e at a concentration of 3 mM caused the decrease of leaves thickness all the species except \u003cem\u003eR. tripserma\u003c/em\u003e which indicatied that \u003cem\u003eR. tripserma\u003c/em\u003e is more tolerant to Hg than the others. Other factors determined leaf thickness may be related to the ability to absorb water, as found by Yao et al. (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) that \u003cem\u003eLycium barbarum\u003c/em\u003e increased their leaf thickness under salt stress to retain water and improve water availability in their leaves. Aside to make duckweeds afloat in water, the frond aerenchyma have crucial function to ensure gas transport in duckweed frond. In these studies, tailings in high concentration caused duckweeds had large aerenchyma in their frond which appears significant in \u003cem\u003eL. aequinoctialis\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee). Kim et al. (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) studies revealed that phytohormone ethylene is responsible for the increasing of aerenchyma areas in duckweed (\u003cem\u003eSpirodela polyrhiza\u003c/em\u003e) by promoting lysigeny via programmed cell death (PCD). Stress including stress by heavy metals could increase the production of ethylene by plants as part of stress response mechanism (Nguyen et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The area of aerenchyma and mesophyll thickness can influence the photosynthetic capacity by changing the mesophyll surface area (Ames/A) (Sack et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). From previous studies by Longstreth and Nobel (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1979\u003c/span\u003e), during salinity stress, plant that reactive to salinity increased their Ames/A by thickening their leaves to conversate stomata closure during stress. Leached tailings containing a lot of soluble solid include heavy metals which can induce salinity and heavy metal stress. Comparing Ames/A from these two species revealed that \u003cem\u003eL. punctata\u003c/em\u003e had better mechanism to counter measured stress by tailings as their Ames/A was relatively constant in all tailings treatment media while high concentration of tailings caused Ames/A of \u003cem\u003eL. aequinoctialis\u003c/em\u003e decrease significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ef).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eEffectiveness of two duckweed species in gold phytomining\u003c/h2\u003e \u003cp\u003eDuckweed (Lemnaceae) has been considered as highly resistance aquatic plant to various pollutants in water, including heavy metals (Golob et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Muthan et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Szab\u0026oacute; et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), which promotes the utilization of duckweed as a heavy metal-absorbing plant for phytoremediation programs. However, there has not been much research that reveals the potential of duckweed in the phytomining of precious metals. The duckweed's ability to absorb large amounts of gold, suggests the potential role of this species in phytomining of gold mine tailings (Yusuf et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Meanwhile, the use of duckweed in phytomining also faces obstacles considering that gold mine tailings contain several heavy metals in high amounts.\u003c/p\u003e \u003cp\u003eBased on gold absorption data, both duckweed species (\u003cem\u003eL. punctata\u003c/em\u003e and \u003cem\u003eL. aequinoctialis\u003c/em\u003e) have the ability to absorb gold very well from gold mine tailings and from gold solution in form HAuCl\u003csub\u003e4\u003c/sub\u003e given in the media, with bioconcentration factor values ​​for gold between 2 and 7, even though the BCF value continued to decrease with increasing tailings concentration. On the other hands, the treatment with addition of HAuCl\u003csub\u003e4\u003c/sub\u003e showed a very high BCF value, i.e.: 131 and 169 for \u003cem\u003eL. aequinoctialis\u003c/em\u003e and \u003cem\u003eL. punctata\u003c/em\u003e respectively (Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The BCF values ​​of these two species are much higher than those obtained by Septian et al. (2025) in \u003cem\u003eAmaranthus spinosus\u003c/em\u003e and \u003cem\u003eBrassica juncea\u003c/em\u003e plants grown in 100% gold mine tailings in combination with ammonium thiocyanate applications. The gold content in both duckweed species was also quite high, i.e.: 0.9\u0026ndash;1.7 mg Kg\u003csup\u003e-1\u003c/sup\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea) and has exceeded the minimum concentration required as a plant for phytomining purposes by Dinh et al. (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), i.e.: at a minimum of 0.06 mg kg\u003csup\u003e-1\u003c/sup\u003e. Among the two duckweed species, gold absorption increased linearly with increasing tailings concentration up to 10%, but only in \u003cem\u003eL. punctata\u003c/em\u003e it still increased at the tailings concentration of 20%, while the absorption in \u003cem\u003eL. aequinoctialis\u003c/em\u003e it was decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea). This condition is in line with the observed physiological parameters, that \u003cem\u003eL. punctata\u003c/em\u003e has higher levels of ascorbate and proline as a sign of a better resistance level than \u003cem\u003eL. aequinoctialis\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ee and h). However, because at a tailings concentration of 20%, both species experienced a decrease in biomass, so that the highest total gold was obtained in the 10% tailings concentration treatment (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Furthermore, gold absorption rate in media with HAuCl\u003csub\u003e4\u003c/sub\u003e solution also suggested that \u003cem\u003eL. punctata\u003c/em\u003e had significantly higher Au absorption rate (23.3 mg Kg\u003csup\u003e-1\u003c/sup\u003e) than \u003cem\u003eL. aequinoctialis\u003c/em\u003e (18 mg Kg\u003csup\u003e-1\u003c/sup\u003e) which indicates a better resistance level (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The decrease in BCF value due to increasing tailings concentration indicates that in addition to the increase of gold content in the media, increasing tailings concentration also means increasing the content of other heavy metals, thereby reducing plant\u0026rsquo;s ability to absorb gold alone. Meanwhile, High levels of heavy metals cause the plants to experience higher stress so that their absorption ability decreases. As a comparison, using Pb treatment, Manzoor et al. (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) found that most of the ornamental plants grown in the highest Pb treatment media (2000 mg kg\u003csup\u003e-1\u003c/sup\u003e) had a lower BCF compared to other treatments with lower Pb concentration.\u003c/p\u003e \u003cp\u003eAnother interesting aspect of these two duckweed species is the formation of gold nanoparticles, which was clearly observed when both species were treated with a 138 ppm Au solution (HAuCl\u003csub\u003e4\u003c/sub\u003e). This was indicated by the formation of a purple color on the fronds of both species (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) and by the purple color of the plant extracts as well as the formation of an absorption peak in the range of 530\u0026ndash;550 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003ea and b). The purple coloration from these two duckweeds did not come from anthocyanin accumulation but from formation of gold nanoparticles inside the plants. This conformed by the purple pigment did not soluble in ethanol solution (used for reducing sugar quantification) which it normally should extract anthocyanins pigment. Our previous studies successfully confirmed the formation of gold nanoparticles in \u003cem\u003eL. aequinoctialis\u003c/em\u003e that grew in Au 138 ppm treatment media using transmission electron microscope (TEM) (Yusuf et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). A similar finding was also demonstrated by Taylor et al. (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) who found that \u003cem\u003eA. thaliana\u003c/em\u003e plants grown in a medium containing gold solution in form of KAuCl\u003csub\u003e4\u003c/sub\u003e changed their leaf color from green to purple, indicating the accumulation of gold nanoparticles in the tissue. Based on the data above (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e), both species produce gold nanoparticles in their tissue after exposing them to media containing HAuCl\u003csub\u003e4\u003c/sub\u003e. The purpose of the 138 ppm Au treatment was to observe the ability of duckweeds to absorb gold without disturbance from other heavy metals. It seems that \u003cem\u003eL. punctata\u003c/em\u003e was superior to \u003cem\u003eL. aequinoctialis\u003c/em\u003e in accumulating gold in their biomass (Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Gold nanoparticles from \u003cem\u003eL. punctata\u003c/em\u003e are predicted to have larger particle sizes compared to \u003cem\u003eL. aequinoctialis\u003c/em\u003e based on the red shift phenomenon (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eb), where the LSPR peak of gold nanoparticles shifts to longer wavelengths as the particles increase in size (Kimling et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). The large size of gold nanoparticles in \u003cem\u003eL. punctata\u003c/em\u003e may be related to their ability to accumulate gold at high concentrations. In the chemical synthesis of gold nanoparticles, the ratio of gold ion to reductant compound plays a crucial factor in determining the size of nanoparticles, which typically have a larger size when the gold ion is more abundant in the reaction (Shi et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The superiority of \u003cem\u003eL. punctata\u003c/em\u003e in gold absorption was in line with physiological changes that can reduce the stress damages characterized by higher ascorbic acid and proline content compared to \u003cem\u003eL. aequinoctialis\u003c/em\u003e. Nevertheless, both have great potential as gold phytomining agents in media containing low gold content, such as tailings.\u003c/p\u003e \u003c/div\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eTwo duckweed species (\u003cem\u003eL. punctata\u003c/em\u003e and \u003cem\u003eL. aequinoctialis\u003c/em\u003e) grown in a medium contained gold mine tailings and gold solution (HAuCl\u003csub\u003e4\u003c/sub\u003e) during the phytomining process experienced stress, resulting in a decrease in growth rate during 7 days of treatment. After the treatment, the frond changed from green to yellow in the tailing\u0026rsquo;s medium treatment, while the frond of duckweeds grown in HAuCl\u003csub\u003e4\u003c/sub\u003e medium turned color to purple. Both the tailings and HAuCl\u003csub\u003e4\u003c/sub\u003e treatments also caused a significant decrease in the frond thickness of the duckweeds. The phytomining process with tailings and HAuCl\u003csub\u003e4\u003c/sub\u003e induced oxidative stress in duckweed, and in response, the plant produced higher ROS-scavenging compounds including proline and reduced sugar. \u003cem\u003eL. punctata\u003c/em\u003e had a higher resistance level to oxidative stress caused by heavy metals compared to \u003cem\u003eL. aequinoctialis\u003c/em\u003e based on morphological and physiological parameters. Both types of duckweed can be categorized as good gold phytomining agents for gold mine waste and can accumulate gold up to 1.7 and 1.5 \u0026micro;g g\u003csup\u003e-1\u003c/sup\u003e for \u003cem\u003eL. punctata\u003c/em\u003e and \u003cem\u003eL. aequinoctialis\u003c/em\u003e, respectively. In Hoagland solution containing 138 ppm Au, gold accumulation by \u003cem\u003eL. punctata\u003c/em\u003e almost reached 2.3% of their biomass, which was significantly more abundant compared to \u003cem\u003eL. aequinoctialis\u003c/em\u003e which was only 1.8%. Gold accumulation by duckweed grown in gold solution (HAuCl\u003csub\u003e4\u003c/sub\u003e) was also high enough to induce the synthesis of gold nanoparticles indicated by purple color of their fronds, that was verified by LSPR measurements showing the formation of peaks at 545 nm and 538 nm for \u003cem\u003eL. punctata\u003c/em\u003e and \u003cem\u003eL. aequinoctialis\u003c/em\u003e, respectively.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThe authors declare that no funds, grants, or other support were received during the preparation of this manuscript.\u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e \n \u003cp\u003eRizki Maulana Yusuf: investigation; methodology; conceptualization; data curation; formal analysis; project administration; writing \u0026ndash; review and editing.\u003c/p\u003e\n\u003cp\u003eAurora Karina Chandra: investigation; methodology; resources; data curation; visualization; formal analysis; project administration.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHamim: supervision; conceptualization; writing \u0026ndash; review and editing; data curation; validation.\u003c/p\u003e\n\u003cp\u003eMiftahudin: supervision; validation; data curation.\u003c/p\u003e\n\u003cp\u003eDorly: supervision; validation; data curation.\u003c/p\u003e\n\u003cp\u003eAwalina Satya: supervision; validation; data curation.\u003c/p\u003e\n\u003cp\u003eEvi Susanti: supervision; validation; data curation.\u003c/p\u003e\n\u003cp\u003eAll authors have read and agreed to the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eACKNOWLEDGEMENT\u003c/h2\u003e \u003cp\u003eThe authors gratefully acknowledge the Research Center for Limnology-National Research and Innovation Agency of the Republic of Indonesia (BRIN) for assistance in analyzing our samples. Also, many thanks to PT. Aneka Tambang (ANTAM Inc.) UBPE Pongkor for supporting research, and all three internal reviewers: Dr. Miftahul Huda Fendiyanto (The Republic of Indonesia Defense University, Bogor, Indonesia), Dr. Sulistijorini (IPB University, Bogor, Indonesia) and Dr. Surono (BRIN, Indonesia) for their valuable time.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbera H, Abdisa M, Washe AP (2020) Spectrophotometric method to the determination of ascorbic acid in \u003cem\u003eM. stenopetala\u003c/em\u003e leaves through catalytic titration with hexavalent chromium and its validation. 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Water Sci Technol 86:2974\u0026ndash;2986. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2166/wst.2022.392\u003c/span\u003e\u003cspan address=\"10.2166/wst.2022.392\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZiegler P, Adelmann K, Zimmer S, Schmidt C, Appenroth KJ (2015) Relative in vitro growth rates of duckweeds (Lemnaceae)\u0026ndash;the most rapidly growing higher plants. Plant Biol 17:33\u0026ndash;41. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/plb.12184\u003c/span\u003e\u003cspan address=\"10.1111/plb.12184\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-and-soil","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plso","sideBox":"Learn more about [Plant and Soil](https://www.springer.com/journal/11104)","snPcode":"11104","submissionUrl":"https://submission.nature.com/new-submission/11104/3","title":"Plant and Soil","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"gold recovery, heavy metal, Lemnaceae, phytoremediation, sustainable mining","lastPublishedDoi":"10.21203/rs.3.rs-9578120/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9578120/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground and aims\u003c/h2\u003e \u003cp\u003ePhytomining can be utilized to extract gold from low-concentration sources like gold mine tailings. Duckweeds (Lemnaceae) have been known as heavy metal hyperaccumulators. This research aimed to observe the morphological, anatomical, and physiological characteristics of two duckweed species (\u003cem\u003eLandoltia punctata\u003c/em\u003e and \u003cem\u003eLemna aequinoctialis\u003c/em\u003e) grown in media containing tailings and gold solution (HAuCl\u003csub\u003e4\u003c/sub\u003e) and their gold accumulating capability.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThe experiment was carried out by exposing \u003cem\u003eL. punctata\u003c/em\u003e and \u003cem\u003eL. aequinoctialis\u003c/em\u003e to 2.5 L of Hoagland\u0026rsquo;s solution treated by 0 g (0%), 100 g (4%), 250 g (10%), 500 g (20%) of tailings suspended by aqua regia, or by 0.7 mM of HAuCl\u003csub\u003e4\u003c/sub\u003e (Au 138 ppm). The treatments and observation were carried out in greenhouse conditions for 7 days. The duckweeds were then harvested for anatomical and physiological measurement, and gold accumulation analysis.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003ehigh content of tailing or Au treatment caused stunting growth in both duckweeds. Anatomical and physiological observations revealed that both duckweeds experienced heavy metal stress, indicated by increasing reactive oxygen species (ROS) and thinner fronds. \u003cem\u003eL. punctata\u003c/em\u003e had superior ability to tolerate the stress based on the ability to increase ROS scavengers to countermeasure ROS production. In addition, under the highest tailing content (20% tailing), \u003cem\u003eL. punctata\u003c/em\u003e was still able to accumulate gold up to 1.7 \u0026micro;g g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and produced larger gold nanoparticles with gold content reached 2.3% in media containing Au 138 ppm.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe gold phytomining process caused stress in duckweeds. Nevertheless, both species have great potential as gold phytomining agents.\u003c/p\u003e","manuscriptTitle":"Morphophysiological Characteristics of Two Duckweed Species Grown in Gold Mine Tailings and HAuCl4 for Phytomining Purpose","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-18 14:34:26","doi":"10.21203/rs.3.rs-9578120/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2026-05-07T13:55:43+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-05-07T13:43:32+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Plant and Soil","date":"2026-05-05T00:04:05+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-05-04T12:19:10+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant and Soil","date":"2026-05-02T09:18:55+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-and-soil","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plso","sideBox":"Learn more about [Plant and Soil](https://www.springer.com/journal/11104)","snPcode":"11104","submissionUrl":"https://submission.nature.com/new-submission/11104/3","title":"Plant and Soil","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"05ba0c00-6c7f-4005-a878-2a8661c07296","owner":[],"postedDate":"May 18th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"","date":"2026-05-07T13:55:43+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-05-07T13:43:32+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-18T14:34:26+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-18 14:34:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9578120","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9578120","identity":"rs-9578120","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}